PT2211901E - Vaccination with multiple clades of h5 influenza a virus - Google Patents

Description

DESCRIPTION

VACCINATION WITH MULTIPLE CLOSURES OF FLU VIRUS H5 A

TECHNICAL FIELD

This invention is in the field of vaccines to protect against infection by the influenza virus, and in particular for protection against influenza pandemics.

STATE OF THE TECHNIQUE

Influenza virus strains for use in vaccines change from season to season. For several years, vaccines have normally included two strains of influenza A (H1N1 and H3N2) and one strain of influenza B. To deal with " antigenic drift ", the exact strains used for vaccination change from year to year.

More troubling than the antigenic drift is the " antigenic shift " in which the prevailing influenza A virus subtype changes from H1N1 and H3N2. In particular, the H5 subtype of the influenza A virus is expected to become prevalent in the near future. As the human population is immunologically weak for the new subtype of hemagglutinin, then this antigenic shift may cause an outbreak of influenza pandemic. Characteristics of a strain of influenza that give it the potential to cause a pandemic are as follows: (a) contain a new hemagglutinin compared to the hemagglutinins currently in circulation in human strains, i.e., one that has not been evident in the population more than a decade, or has not been seen in the entire human population; (b) be able to be transmitted horizontally in the human population; and (c), be pathogenic to humans. 1

In preparation for a pandemic influenza caused by an H5 strain, a pre-pandemic vaccination strategy has been proposed [1]. Patients are immunized with the current H5 (bird) strain in the hope that the resulting immunity will be useful when the pandemic occurs, although any antigenic drift may occur in the same period.

In October 2006, Sanofi Pasteur announced the results of its candidate pre-pandemic vaccine, which was tested in adjuvant and non-adjuvant forms at various doses of antigen. In November 2006 Novartis Vaccines announced that a European regulation for an adjuvant vaccine against pre-pandemic has been submitted for approval. In June 2007 GlaxoSmithKline announced its intention to donate 50 million doses of adjuvant vaccine against the WHO H5N1 pre-pandemic vaccine and in February 2008 its PREPANDRIX ™ product received a positive opinion from the EMEA Committee for Medicinal Products for Human Use. All three products were based on strain H5N1 / A / Vietnam / 1194/04.

Vaccine, Vol. 21, 2003, 1687-1693 discloses the administration of an H5N3-based vaccine to increase immunity to the H5N1 virus.

DISCLOSURE OF THE INVENTION H5N1 virus isolated from animals and humans since 2003 separated into several distinct clades (gene groups) of closely related viruses based on hemagglutinin amino acid sequences. The strain H5N1 / A / Vietnam / 1194/04 is classified in cledo 1. 2

According to the invention, as defined in the claims, multiple clades are used in the immunization against influenza.

In a first aspect of the invention (see claim 1), a first boost immunization scheme is used when a subject receives a preparation dose of a first clade of influenza A virus H5 and a boost dose of a second virus clade influenza A H5. For example, one patient may start with clade I antigen and strengthen with clade 2, or vice versa.

In the same way (see claim 2), a booster immunization can be administered to a patient who has already received an H5 vaccine from a first clade (e.g. clade 1) through an H5 vaccine from a different clade (for example, clade 2). The patient generally receives a complete primary sequence (for example, two doses) of the first clade of the vaccine.

In a second aspect, an immunogenic composition may comprise hemagglutinin antigens from various clades of the H5 influenza A virus, allowing simultaneous immunization against multiple H5 clades.

Clades of H5

The hemagglutinin antigens of the viruses used with the invention fall into the H5 subtype, but within the H5 subtype they fall into different clades.

The hemagglutinin (HA) sequences of most H5N1 viruses circulating in bird species since 2003 are separated into distinct phylogenetic clades. Clade I virus circulating in Cambodia, Thailand and Vietnam was responsible for human infections in these countries in 2004 and 2005, and Thailand in 2006. Clade II virus has been circulating in poultry in China and Indonesia since 2003; and spread westward throughout 2005 and 2006 to the Middle East, Europe and Africa. Since the end of 2005, clade 2 virus has been primarily responsible for human infection. Several subclades of clade 2 have been distinguished: three of them - subclades 1, 2 and 3 (Fig. 1) - differ in geographic distribution and so far have been largely responsible for cases in humans.

Between August 2006 and March 2007, most of the H5N1 HA sequences that have continued to circulate or resurged in bird species and have been associated with sporadic human infections in Africa, Asia and Europe belong to the previously designated phylogenetic clades and subclades. Clade I virus was responsible for outbreaks in birds in Thailand and Vietnam and human infections in Thailand. Clade 2.1 virus continues to circulate in poultry and to cause human infections in Indonesia. Clade virus 2.2 has caused outbreaks in birds in some countries in Africa, Asia and Europe and has been associated with human infections in Egypt, Iraq and Nigeria. Clade 2.3 virus has been sporadically isolated in Asia and has been responsible for human infections in China and the Lao People's Democratic Republic.

In addition, some viruses outside these classifications have been isolated in poultry during outbreaks in Asia. These are divided into emergent clades, represented by A / goose / Guiyang / 337/2006 4 (clade 4) and A / chicken / Shanxi / 2/2006 (clade 7). In total, 10 clades have now been defined (Figure 2), numbered from 0 to 9.

For reference, prototypic strains for each clade are as follows, together with the coding sequence of the hemagglutinin genes:

Clade Strain SEQ ID No. 1 A / HongKong / 213/03 1 2 A / Indonesia / 5/05 2 3 A / Chicken / Hong Kong / SF219 / 01 3 4 A / Chicken / Guiyang / 441/2 0 0 6 4 5 A / Duck / Guangxi / 1681/2004 5 6 A / Chicken sparrow / Henan / 4/2004 6 7 A / Chicken / Shanxi / Guangxi / 2775/2005 9 0 A / Hong Kong / 156/97 10

An H5 virus from clade 1 can be defined phylogenetically as an influenza A virus having a sequence encoding the hemagglutinin which is most closely related to the coding sequence from strain A / HongKong / 213/03 (SEQ ID NO: No. 1) than with any coding sequence of any of clades 0 and 2 to 9 (SEQ ID NOs: 2-10), when evaluated using the DNADIST algorithm as implemented in the Phylip [2] package (e.g. , using two Kimura distance parameters and a square matrix). Likewise, a clade 2 virus has a hemaqlutinin coding sequence that is most closely related to the coding sequence from strain A / Indonesia / 5/05 (SEQ ID NO: 2) than with any sequence coding of any of the clades 0, 1 and 3 to 9 (SEQ ID NOS: 1 and 3 to 10). The other clades are phylogenetically defined in the same way - with a hemaqlutinin coding sequence which is most closely related to the relevant coding sequence from SEQ ID NOS: 1 to 10 than with the other sequences in SEQ ID NO: ° s: 1 to 10.

A clade 1 virus can be defined herein in terms of the nucleic acid sequence as a virus of strain A having a hemagglutinin coding sequence with a higher sequence identity to strain A / HongKong / 213/03 (SEQ ID NO: 1) than any of SEQ ID NOs: 2 and 10. The other clades are defined in the same way - with a hemagglutinin coding sequence which is most closely related to the relevant coding sequence from the SEQ ID Nos: 1 to 10 than for the other sequences in SEQ ID Nos: 1 to 10.

An H5 virus can be defined herein as being a particular clade, in terms of amino acid sequence, by reference to characteristic HA mutations [3]. For example, a clade 3 virus may have one or more of the following amino acid residues, which are distinct from clades 1 and 2: Asn-45; Ser-84; Asn-94; Asn-124; Leu-138;

Ser-144; Glu-212; Ser-223; and / or Arg-325. A clade virus may have Asp-124, which is not seen in clades 1 and 3. A clade virus 1 may have one or more of the following amino acid residues 6, which are distinct from clades 2 and 3: Ser-124; Leu-129.

In clade 2, at least three subclasses were recognized: 2.1, 2.2 and 2.3 (Figure 1). A H5 clade 2.1 virus may be defined phylogenetically herein as having a hemagglutinin coding sequence which is more closely related to strain A / Indonesia / 5/05 (SEQ ID NO: 2) than for both the strain A / Anhui / 1/2005 (SEQ ID NO: 11) or A / turkey / Turkey / 1/05 (SEQ ID NO: 12). Likewise, a H5 2.2 clade virus can be defined phylogenetically as having a hemagglutinin coding sequence which is most closely related to strain A / turkey / Turkey / 1/05 (SEQ ID NO: 12) (SEQ ID NO: 11) and strain A / Indonesia / 5/05 (SEQ ID NO: 2). Finally, a H5 clade 2.3 virus may be defined phylogenetically as having a hemagglutinin coding sequence which is more closely related to strain A / Anhui / 1/05 (SEQ ID NO: 11) than both the strain A / turkey / Turkey / 1/05 (SEQ ID NO: 12) or strain A / Indonesia / 5/05 (SEQ ID NO: 2).

In some embodiments a strain in subclone 2.2 may have HA, including one or more of the following sequences: Ile-223; Ile-230; Ser-294; Ile-517; ASer-133; a cleavage site having the sequence REGRRRKR (SEQ ID NO: 13), a cleavage site with the sequence GERRRRKR (SEQ ID NO: 16). The HA gene may include one or more of the nucleotides: A-41; A-142; A-209; A-295; G-433; A-467; A-496; C-610; A-627; A-643; C-658; T-661; T-689; T-727; A-754; G-880; C-937; G-1006; T-1012; A-1019; T-1177; A-1235; T-1402; C-1415; T-1480; C-1510; T-1614; C-1615; A-1672; G-1708 (either of which may or may not alter the 7 amino acid encoded by the relevant codon). The NA gene may include nucleotides A-743, which will not alter the amino acid encoded by the relevant codon.

In some embodiments of the invention, the immunizing antigen and / or the boosting antigen are not of the clade, for example, they are not from strain A / HongKong / 156/97. For example, if the second antigen is from clade 1 (A / Vietnam / 1203/2004 for example), then the first antigen is not preferably clade 0 (for example, A / Hong Kong / 156/97).

Clade Combinations The invention utilizes antigens derived from more than one H5 clade for immunization.

For example, a patient may be prepared with the antigen of an H5 virus in a first clade, but then carry a boost with the antigen of an H5 virus in a second clade. Useful clade combinations in this initiation / reinforcement strategy include, but are not limited to, the following:

Preparation 1 2 1 1 2 2 1 1 1 2.1 2.2 2.3 Reinforcement 2 1 4 7 4 7 2.1 2.2 2.3 1 1 1 The invention also provides in claim 2 booster immunization for a patient previously immunized with antigen of an H5 virus in a first clade , where the reinforcement is of a second (different) clade. Combinations 8 above may also be used in this initiation / reinforcement approach. The invention also provides in claim 8 an immunogenic composition comprising H5 virus antigens in at least two different clades. Useful combinations of two clades include, but are not limited to:

First 1 1 1 1 1 1 1 1 2 2 1 1 1 Second 2 3 4 5 6 7 8 9 4 7 2.1 2.2 2.3 0 Influenza virus antigen

The vaccines used with the invention include an influenza virus antigen having a H5 hemagglutinin subtype. The vaccine will comprise a protein comprising at least one epitope of hemagglutinin H5, for example, the vaccine will comprise viral hemagglutinin of an H5 virus.

Preferred vaccines of the invention also include a protein comprising at least one influenza virus neuraminidase epitope, for example the vaccine includes the viral neuraminidase of an H5 virus. The invention may protect against one or more of the subtypes of influenza A virus N1, N2, N3, N4, N5, N6, N7, N8, N8 or N9 but will generally be against N1 (i.e., a H5N1 virus) or N3 (ie an H5N3 virus). The first and second clade may have the same subtype of NA (for example, both N1 or both N3), or different subtypes of NA (e.g. N1, then N3, or N3 and then N1, etc.) . If only one of the preparation and booster doses includes neuraminidase, it may be the dose of preparation. The antigen will typically be prepared from influenza virions, but, alternatively, antigens, such as hemagglutinin and neuraminidase, may be expressed in a recombinant host (e.g., an insect cell line using a baculovirus vector) and used in the purified form [4, 5, 6], or in the form of virus-like particles (VLPs, for example, see references 7 and 8). In general, however, the antigens will be from virions.

The antigens may take the form of a live virus or, more preferably, an inactivated virus. Chemical means for inactivating a virus include treatment with an effective amount of one or more of the following agents: detergents, formaldehyde, formalin, β-propiolactone, or UV light. Additional means for chemical inactivation include treatment with methylene blue, psoralen, carboxyferene (C60), or a combination of any of these. Other methods of viral inactivation are known in the art, such as, for example, binary ethylamine, ethyleneimine acetyl or gamma irradiation.

Where an inactivated virus is used, the vaccine may comprise an entire virion, a fragmented virion or purified surface antigens (including haemagglutinin and usually also including neuraminidase). The product H5N1 DARONRIX ™ is an entire inactivated virion vaccine. The product H5N1 PREPANDRIX ™ is an inactivated fragmented virion vaccine. The fragmented virion and the purified surface antigens (i.e., subvirus vaccines) are particularly useful with the invention. 10

Virions can be harvested from fluids containing viruses by various methods. For example, a purification process may involve zonal centrifugation using a linear sucrose gradient solution which includes a detergent to break the virions. The antigens may then be purified, after optional dilution, by diafiltration.

Fragmented virions are obtained by treating the virions with detergents (eg, ethyl ether, polysorbate 80, deoxycholate, tri-n-butyl phosphate, Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9, etc.) for produce subvibry preparations, including the " Tween-ether " split process. Methods of separating influenza virus are well known in the art see, for example, References 9-14, etc. Division of the virus is usually accomplished by rupturing or fragmenting the entire virus, whether infectious or non-infectious, with a breaking concentration of a cleaving agent. Rupture results in complete or partial solubilization of virus proteins, altering the integrity of the virus. Preferred splitting agents are nonionic and ionic (e.g. cationic) surfactants, for example, alkylglycosides, alkylthioglycosides, acyl sugars, sulfobetaines, betaines, polyoxyethylene alkyl ethers, N, N-dialkylglucamides, Hecameg, alkylphenoxypolyethoxyethanols, quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-n-butyl phosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin, lipofectamine, and DOT-MA, octyl or nonylphenoxy polyoxyethanols (for example surfactants Triton, such as Triton X-100 or Triton N101), polyoxyethylene sorbitan esters (Tween surfactants), polyoxyethylene ethers, polyoxyethylene esters, etc. A useful cleavage procedure utilizes the consecutive effects of sodium deoxycholate and formaldehyde, and cleavage may take place during the initial purification of the virion (for example, in a sucrose density gradient solution). Thus, a cleavage process may involve clarification of the virion-containing material (to remove non-virion material), concentration of the virions harvested (for example, using an adsorption method, such as adsorption by CaHP04), separation of virions from non-virion material, dividing the virions using a separating agent, in a density gradient centrifugation step (for example, using a sucrose gradient containing a separating agent, such as sodium deoxycholate) , and then filtration (e.g., ultrafiltration) to remove undesired materials. Virion division may be useful by resuspension in isotonic solution of sodium chloride in sodium phosphate buffer. BEGRIVAC ™, FLUARIX ™, Fluzone ™ and FLUSHIELD ™ are split vaccines.

Purified surface antigen vaccinates comprise the influenza hemagglutinin surface antigens and usually also the neuraminidase. Processes for the preparation of such proteins in the purified form are well known in the art. Fluvirin ™, CHIROFLU ™, FOCETRIA ™ and Influvac ™ are subunit vaccines.

Influenza antigens may also be presented as virosomes [15] (nucleic acid free of viral particles, such as liposomes), as in INFLEXAL V ™ and INVAVAC ™ products, but it is preferable not to use virosomes with the present invention. Thus, in some embodiments, the influenza antigen is not in the form of a virosome. The strain from which the virus is prepared will typically be an avian influenza virus or a human influenza virus. It will generally be able to infect humans, but in some cases a strain that can be used can not infect humans, for example a strain of bird flu that may later acquire the ability to infect humans. The strain may be a strain of HPAI (highly pathogenic avian influenza) [16]. The influenza virus can be attenuated. Influenza virus may be temperature sensitive. The influenza virus can be adapted to cold. These three characteristics are particularly useful when using the live virus as an antigen. Flu viruses may be resistant to antiviral therapy (eg, oseltamivir resistant [17] and / or zanamivir).

The compositions of the invention may include the antigen (s) of one or more (e.g., 1, 2, 3, 4 or more) strains of influenza virus, including influenza A virus and / or influenza B virus, provided they include the antigen of at least one H5 strain. Thus, a composition may include the antigen of one or more characteristics of the strain of a normal seasonal vaccine and of at least one H5 strain, for example a valence 4 vaccine with three strains of influenza A virus (H1N1 and H3N2, in addition of an H5 strain eg 13.5æ1), and an influenza B strain. In other embodiments, the antigen of at least two H5 strains and optionally a H1 strain and / or an H3 strain and / or a strain of influenza B virus. When a vaccine includes more than one influenza strain, the different strains are usually cultured separately and are mixed after the virus has been harvested and the antigens prepared. Thus, a process of the invention may include the step of blending antigens from more than one influenza strain. Influenza virus may be a recombinant strain, and may have been obtained by reverse genetics techniques. Reverse genetics techniques [e.g., 18-22] allow influenza viruses with segments of the desired genome to be prepared in vitro using plasmids. Typically, it involves expressing (a) DNA molecules encoding the desired viral RNA molecules, for example, from individual promoters, and (b) DNA molecules encoding viral proteins, for example from individual promoters, in such a way that the expression of both types of DNA in a cell leads to the assembly of an intact complete infectious virion. DNA preferably provides all viral RNAs and proteins, but it is also possible to use a helper virus to provide some of the RNA and proteins. Plasmid-based methods are preferred, using separate plasmids for the production of each viral RNA [23-25], and these methods may also involve the use of plasmids to express all or part (for example, only PB1, PB2, PA and NP) of the viral proteins, with up to 12 plasmids to be used in certain methods. If canine cells are used, a single canine promoter can be used [26]. 14

To reduce the number of plasmids required, an approach [27] combines a plurality of RNA polymerase I transcription cassettes (for viral RNA synthesis) on the same plasmid (e.g., sequences encoding 1, 2, 3, 4 , 5, 6, 7 or all 8 influenza A vRNA segments), and a plurality of protein coding regions with RNA polymerase II promoters on another plasmid (e.g., sequences encoding 1, 2, 3, 4 , 5, 6, 7 or all 8 influenza A mRNA transcripts).

Preferred aspects of the reference method 27 involve: (a) coding regions of PB1, PB2 and PA mRNA in a single plasmid; and (b) all 8 coding segments of vRNA in a single plasmid. Including the NA and HA segments on one plasmid and the other six segments on another plasmid may also facilitate the materials.

As an alternative to the use of individual promoters to encode the viral RNA segments, it is possible to use the bacteriophage polymerase promoters [28]. For example, promoters for the SP6, T3 or T7 polymerases can be conveniently used. Due to the specificity of individual promoter species, bacteriophage polymerase promoters may be more convenient for many cell types (e.g. MDCK), although a cell must also be transfected with a plasmid encoding the exogenous polymerase enzyme.

In other techniques, it is possible to use single and double single promoters encoding both viral RNAs and expressible mRNAs from a single template [29,30]. Influenza A virus may comprise one or more RNA segments of an A / PR / 8/34 virus (typically 6 segments of A / PR / 8/34, with the HA and N segments being of a vaccine strain, that is, a 6: 2 rearrangement), especially when viruses are produced in eggs. It may also include one or more RNA segments of an A / WSN / 33 virus, or any other virus strain useful for generating recombinant viruses for the preparation of the vaccine. Typically, the invention protects against a strain that is capable of human to human transmission, and thus the strain genome generally includes at least one RNA segment that originated from a mammalian (e.g., a human) influenza virus. It may include the NS segment that originated from a bird flu virus.

Viruses used as the source of the antigens can be cultured in the eggs or in cell culture. The current standard method for the growth of the influenza virus uses specific pathogen free embryonic hen eggs (SPF), with the virus being purified from the contents of the eggs (allantoic fluid). More recently, however, viruses have been produced in animal cell culture and, for reasons of speed and allergies of the patient, this growth method is preferred. If egg-based viral growth is used, then one or more amino acids can be introduced into the egg's allantoic fluid along with the virus [14].

When the cell culture is used, the viral growth substrate will typically be a cell line of mammalian origin. Suitable mammalian origin cells include, but are not limited to, hamster cells, cattle, primates (including humans and monkeys) and dogs. Various types of cells, such as kidney cells, fibroblasts, retinal cells, lung cells, etc. may be used. Examples of suitable hamster cells are the cell lines with the names BHK21 or HKCC. Suitable monkey cells are, for example, African green monkey cells, such as kidney cells as in the Vero cell line. Suitable dog cells are, for example, kidney cells, such as in the MDCK cell line. Thus, suitable cell lines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; etc. Preferred mammalian cell lines for influenza virus culture include: MDCK cells [31-34], derived from the canine kidney Madin Darby; Vero cells [35-37], derived from the African green monkey kidney (Cercopithecus aethiops); or PER.C6 cells [38], derived from human embryonic retinoblasts. These cell lines are widely available, for example from the American Type Cell Culture (ATCC) collection, from Coriell Cell Repositories or from the European Collection of Cell Cultures (ECACC). For example, ATCC provides several different Vero cells, with the accession numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and provides MDCK cells with the CCL-34 catalog number. PER.C6 is available from the ECACC under deposit number 96022940. As a less preferred alternative for mammalian cell lines, viruses can be cultured in avian cell lines [e.g. references 39-41], including cell lines derived from ducks (e.g., duck retina) or chickens. Examples of bird cell lines include avian embryonic stem cells [39,42] and duck retinal cells. [40] Suitable avian embryonic stem cells include the EBx cell line derived from embryonic stem cells from chicken, EB45, EB 14, and 17 ΕΒ14-074 [43]. Chicken embryo fibroblasts (CEF) may also be used.

The most preferred cell lines for culturing the influenza virus are MDCK cell lines. The original MDCK cell line is available from ATCC as CCL-34, but the derivatives of this cell line can also be used. For example, reference 31, discloses an MDCK cell line, which has been adapted for growth in suspension culture (" MDCK 33016 ", deposited as DSM ACC 2219). Also, reference 44 describes a derivative MDCK cell line which grows in serum-free suspension culture (" B-702 ", deposited as FERM BP-7449). Reference 45 discloses non-tumorigenic MDCK cells, including " MDCK-S " (ATCC PTA-6500), " MDCK-SF101 " (ATCC PTA-6501), " MDCK-SF102 " (ATCC PTA-6502) and " MDCK-SF103 " (PTA-6503). Reference 46 describes MDCK cell lines with high susceptibility to infection, including cells " MDCK.5F1 " (ATCC CRL-12042). Any of these MDCK cell lines can be used.

When the virus has been cultured in a mammalian cell line, then the composition will advantageously be free of egg proteins (e.g., ovalbumin and ovomucoid) and chicken DNA, thereby reducing allergic factors.

When the virus has been cultured in a cell line, then the culture for growth, and also the viral inoculum used to start the culture, will preferably be free from (i.e., tested for and given a negative contamination result for example, herpes simplex virus, respiratory synovial virus, paraglottic virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, circovirus, and / or parvovirus 47. The absence of the herpes simplex virus is particularly preferred.

For growth of a cell line, such as in MDCK cells, viruses can be cultured in cells in suspension [48-50] or in adherent culture. One MDCK cell line suitable for suspension culture is MDCK 33016 (deposited as DSM ACC 2219). As an alternative, the micro-carrier culture can be used.

Cell lines which support replication of the influenza virus are preferably cultured in serum free culture medium and / or protein free medium. A medium is referred to as a serum free medium, in the context of the present invention, when there are no serum additives of human or animal origin. Free proteins are understood to be cultures in which cell multiplication occurs with exclusion of proteins, growth factors, other protein additives and non-serous proteins, but may optionally include proteins such as trypsin or other proteases which may be necessary for viral growth. Cells growing in cultures naturally contain proteins themselves.

Cell lines that support influenza virus replication preferably grow below 37øC [51] during viral replication, for example 30-36øC. The method for virus propagation in cell culture in general includes the steps of inoculating the culture cells with the strain to be cultured, culturing the infected cells for a desired period of time for virus propagation, such as, for example, as determined by the virus titer or the expression of the antigen (e.g., between 24 and 168 hours post-inoculation) and collection of the propagated virus. The culture cells are inoculated with a virus (measured by UFP or TCID50) in a cell ratio of 1: 500 to 1: 1, preferably 1: 100 to 1: 5, more preferably 1: 50-1: 10. The virus is added to a cell suspension or is applied to a cell monolayer, and virus is absorbed into the cells for at least 60 minutes, but usually less than 300 minutes, preferably between 90 and 240 minutes at 25 ° C at 40øC, preferably 28øC to 37øC. Infected cell culture (e.g., monolayers) can be removed by freeze-thawing, or by enzymatic action to increase the viral content of harvested culture supernatants. The collected fluids are then inactivated or stored frozen. The cultured cells may be infected with a multiplicity of infection (" moi ") of about 0.0001 to 10, preferably 0.002 to 5, more preferably 0.001 to 2. Even more preferably, the cells are infected at a moi of about 0.01. Infected cells can be harvested 30 to 60 hours after infection. Preferably, the cells are harvested 34-48 hours after infection. Even more preferably, the cells are harvested 38 to 40 hours post-infection. Proteases (trypsin usually) are generally added during cell culture to allow viral release, and the proteases can be added at any suitable stage during the culturing.

Hemagglutinin (HA) is the major immunogen in inactivated influenza vaccines, and vaccine doses are standardized by reference to HA levels, usually measured by single radial immunodiffusion (SRID) assay. Current vaccines typically contain about 15pg of HA per strain, although lower doses are also used, for example in children, or in pandemic situations. Fractionated doses have been used such as (i.e. 7.5 pg HA per strain, as in FOCETRIA ™), H (i.e. 3.75 pg per strain, as in PREPANDRIX ™) and [52,53], as well as higher doses (e.g., 3x or 9x doses [54, 55]). Thus, the vaccines may comprise between 0.1 and 150æg HA per influenza strain, preferably between 0.1 and 50æg, e.g., 0.1-20æg, 0.1-15æg, 0.1æg 0.1æg, 0.5-5æg, etc. Particular doses include, for example, about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9 , about 1.5, etc. pg per strain. An identical mass of HA per strain is typical. For compositions, including HAs of strains of different H5 clades, it is useful to include 3.75 pg, 7.5 pg or 15 pg per strain (for example, 2.times.3.75 pg, giving a total of 7.5 pg of HA, usually in combination with an adjuvant such as a squalene-in-water emulsion). The lower doses (i.e. <15pg / dose) are most useful when an adjuvant is present in the vaccine, as in the invention. Although doses as high as 90æg have been used in some studies (e.g., reference 56), the compositions of the invention (particularly at the booster dose) usually include 15æg / dose or less.

For live vaccines, the dosage is measured by the median infectious dose of tissue culture (TCID50) instead of the HA content and a TCID50 is typically between 106 and 108 (preferably 106.5-107.5) per strain. 21 HA used with the invention is a natural HA, as found in a virus, or may have been modified. For example, it is known that to modify HA to remove determinants (eg, hyper-base regions around the cleavage site between HA1 and HA2) that make a virus highly pathogenic in bird species, since these determinants may possibly prevent a virus from being grown in eggs.

The compositions of the invention may include detergent, for example a polyoxyethylene sorbitan surfactant ester (known as &quot; Tweens &quot;, for example, polysorbate 80), an octoxynol (such as octoxynol-9 (Triton X-100) or 10, or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide ('CTAB'), or sodium deoxycholate, in particular for a division or vaccine of the surface antigen. The detergent may be present only in trace amounts. Thus, the vaccine may comprise less than 1mg / ml of each octoxynol-10, hydrogen α-tocopherol succinate and polysorbate 80. Other trace residual components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B). Host cell DNA

When the virus has been cultured in a cell line, it is standard practice to minimize the residual amount of DNA from the cell line at the end of the vaccine, in order to minimize any oncogenic DNA activity. Thus, when the virus has been cultured in a cell line, the composition preferably contains less than 10 ng (preferably less than, and more preferably less than 100pg) residual host cell DNA per dose, although 22 trace elements of host cell DNA may be present. It is preferred that the average length of a host cell residual DNA is less than 500bp, for example less than 400bp, less than 300bp, less than 200bp, less than 100bp, etc. In general, host cell DNA that it is desirable to exclude in the compositions of the invention is DNA that is greater than 100 bp. Residual DNA measurement of the host cell is now a routine regulatory requirement for biological agents and is within the normal capabilities of the person skilled in the art. The assay used to measure DNA will typically be a validated assay [57,58]. The performance characteristics of a validated assay can be described in mathematical and quantifiable terms, and its possible sources of error have been identified. The assay was generally tested for characteristics, such as accuracy, precision, specificity. Once calibrated assay (for example, against known standard amounts of host cell DNA), and then tested, quantitative DNA measurements can be performed routinely. Three major techniques for DNA quantification may be used: hybridization methods, such as &quot; Southern blot &quot; or &quot; slot blot &quot;[59]; immunoassay methods such as the Threshold System [60], and quantitative PCR [61]. These methods are all familiar to the person skilled in the art, although the exact characteristics of each method may depend on the host cell in question, for example, the choice of probes for hybridization, the choice of primers and / or probes for amplification, etc. . The Threshold ™ system from Molecular Devices is a quantitative assay for total DNA levels in picograms and has been used to monitor levels of DNA contamination in biopharmaceuticals [60]. A typical assay 23 involves non-specific sequence formation of a reaction complex between a biotinylated ssDNA binding protein, a conjugated anti-ssDNA urease antibody and DNA. All components of the assay are included in the Total DNA Assay Kit available from the manufacturer. Several commercial manufacturers offer quantitative PCR assays for residual DNA detection from the host cell eg AppTec ™ Laboratory Services, BioReliance ™, Althea Technologies, etc. A comparison between a chemiluminescent hybridization assay and the host cell contamination measurement system by whole DNA from a Threshold ™ human viral vaccine can be found in reference 62. Contaminant DNA can be removed during the preparation of the vaccine using purification procedures for example chromatography, etc. Removal of the DNA residue from the host cell can be improved by nuclease treatment, for example using a DNase. A convenient method for reducing host cell DNA contamination is described in references 63 and 64, involving a two-step treatment, first using a DNase (for example Benzonase), which can be used during viral growth, and , then a cationic detergent (e.g., CTAB), which may be used during the interruption of the virion. Treatment with an alkylating agent, such as β-propiolactone, can also be used to remove DNA from the host cell, and, advantageously, can also be used to inactivate the virions [65].

Vaccines containing <10ng (eg <100ng) of host cell DNA per 15æg of hemagglutinin are preferred, as are vaccines containing <10ng (eg <100ng) DNA of the host cell per volume of 0.25 ml. Vaccines containing <10ng (eg <100ng) of host cell DNA per 50æg of hemagglutinin are most preferred, as are vaccines containing <10ng (eg <100ng) DNA of the host cell per 0.5 ml volume.

Adjuvant (s)

A composition of the invention may include an adjuvant for improving the immune (humoral and / or cellular) responses produced in a patient receiving the composition. It is preferred that the clade of the first vaccine and the clade of the second vaccine are vaccines with adjuvants. They may use the same adjuvant or different adjuvants. If only one of the two vaccines has adjuvant, it is preferably the second vaccine.

Adjuvants suitable for use with the invention include, but are not limited to: A mineral composition containing, including calcium salts and aluminum salts (or mixtures thereof). Calcium salts include calcium phosphate (for example, the &quot; CAP &quot; particles disclosed in Reference 66). The aluminum salts include hydroxides, phosphates, sulfates, etc., with their salts, having any suitable form (for example, gel, crystalline, amorphous, etc.). Adsorption to these salts is preferred. The compositions containing the mineral may also be formulated as a metal salt particle [67]. Aluminum salt adjuvants are described in more detail below. • An oil-in-water emulsion, as described in more detail below. An immunostimulatory oligonucleotide, as described in more detail below. • 3-0-monophosphoryl deacylated lipid A (&quot; 3 DMPL &quot; also known as &quot; MPL ™ &quot;), as described in more detail below. An imidazoquinoline compound, such as Imiquimod (&quot; R-837 &quot;) [68,69], Resiquimod (&quot; R-848 &quot;) [70], and the analogues thereof; and salts thereof (for example, hydrochloride salts). Further details on immunostimulating imidazoquinolines can be found in references 71 to 75. A thiosemicarbazone compound, such as those described in reference 76. Methods for formulating, manufacturing, and screening active compounds are also described in reference 76. thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α. • An analogous nucleoside, such as: (a) Isatorabin (ANA-245, 7-thia-8-oxoguanosine):

26 and prodrugs thereof, (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds described in references 77 to 79, (f) a compound having the formula:

wherein: R 2 and R 2 are each independently H, halo, -NR a R b, -OH, C 1-6 alkoxy, substituted C 1-6 alkoxy, heterocyclyl, substituted heterocyclyl, C 6-10 aryl, substituted Ceyl, C 1-6 alkyl , or substituted C1-6 alkyl; R3 is absent, H, C1-6 alkyl, substituted C1-6 alkyl, Cg-10 aryl, substituted C6-10 aryl, substituted heterocyclic or heterocyclic; R4 and R5 are each independently H, halo, heterocyclyl, substituted heterocyclyl, -C (O) -Rd, C1-6 alkyl, substituted C1-6 alkyl, or joined together to form a 5-membered ring such as in R4-5:

the connection being reached at the connections indicated by

X 1 and X 2 are each independently N, C, O or S; R8 is H, halo, -OH, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -NRa Rb, - (CH2) n O-Rc, -o- (C1-6 alkyl), -S (O) pRe or -C (O) -R d; R9 is H, C1-6 alkyl, substituted C1-6 alkyl, heterocyclyl, substituted heterocyclyl or Rg, wherein Rg is:

the connection to be reached at the connection indicated by

R10 and R11 are each independently H, halo, C1-6 alkoxy, substituted C1-6 alkoxy, -NRa Rb or -OH; each Ra and Rb is, independently, H, C1-6 alkyl, substituted C1-6 alkyl, -C (O) R5, C6-10 aryl; each R c is independently H, phosphate, diphosphate, triphosphate, C 1-6 alkyl, or substituted C 1-6 alkyl; each R d is independently H, halo, C 1-6 alkyl, C 1-6 substituted alkyl, C 1-6 alkoxy, substituted C 1-6 alkoxy, -NH 2, -NH (C 1-6 alkyl), -NH (substituted C 1-6 alkyl), -N ) 2R -N (substituted alkyl 0-ε) 2, aryl ε-ιο, or heterocyclyl; each R e is independently H, C 1-6 alkyl, C 1-6 substituted alkyl, substituted C 6-9 cycloalkyl, substituted heterocyclic or heterocyclic aryl; Each R f is independently H, C 1-6 alkyl, C 1-6 substituted alkyl, -C (O) R d, phosphate, diphosphate or triphosphate; each n is independently 0, 1, 2, or 3; each p is independently 0, 1 or 2; or (g) a pharmaceutically acceptable salt of any one of (a) to (f), a tautomer of any of (a) to (f), or a pharmaceutically acceptable salt of the tautomer. A compound of triftantrine such as those described in reference 80. Methods of formulating, manufacturing, and screening active compounds are also described in reference 80. Thiosemicarbazones are particularly effective in stimulating human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α. • Loxoribine (7-allyl-8-oxoguanosine) [81]. The compounds disclosed in reference 82, including: Acclpiperazine compounds, Indoledione compounds, Tetrahydrazoisoquinoline compounds (THIQ), Benzocyclodione compounds, Aminoazavinyl compounds, aminobenzimidazole quinolinone compounds (ABIQ) [83,84], Hydrazaphthalamide compounds, benzophenone compounds, compounds of

Isoxazole compounds, Esterol compounds, Quinazilinone compounds, Pyrrole compounds [85], Antraquinone compounds, Quinoxaline compounds, Compounds of

Triazin, Pyrazalopyrimidine Compounds and Benzazole Compounds [86]. The compounds disclosed in reference 87, including 3,4-di- (1H-indol-3-yl) -1H-pyrrole-2,5-diones, staurosporine analogs, derivatized pyridazines, chromen-4-ones, indolinones , quinazolines and nucleoside analogs. • A glucosaminide aminoalkyl phosphate derivative such as RC-529 [88,89]. • A phosphazene, such as poly [di (carboxylatophenoxy) phosphazene] (&quot; PCPP &quot;) as described, for example, in references 90 and 91. • Small immunopotentiating molecules (SMIPs), such as N2-methyl- 2-methylpropyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine N2, N2-dimethyl-1- (2-methylpropyl) imidazo [4,5- c] quinoline- 2-methylpropyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine N2-methyl-1- (2-methylpropyl) -N2- propyl-1H-imidazo [4,5-c] quinoline-2,4-diamine 1- (2-methylpropyl) -N2-propyl- (2-methylpropyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine N2 -butyl-N2-methyl-1- (2-methylpropyl) -1H- (2-methylpropyl) -N2-pentyl-1H-imidazo [4,5-c] quinoline-2,4-diamine N2-methyl-1 (2 2-enyl-1H-imidazo [4,5-c] quinoline-2,4-diamine 1- (2-methylpropyl) -2 - [(phenylmethyl) thio] -1H-imidazo [ 4,5-c] quinoline-4-amine 1- (2-methylpropyl) -2- (propylthio) -1H-imidazo [4,5-c] quinoline (2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl] (methyl) amino] ethanol 2 - [[4-amino Ethyl 4-amino-1- (2-methylpropyl) -1,3-dihydro-1H-imidazo [4,5- c] quinolin-2-yl] 2-one N 2 -butyl-1- (2-methylpropyl) -N 4, N 4 -bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine N2 -butyl-N2-methyl- methyl-1- (2-methylpropyl) -N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine N2, N2-dimethyl-1- (2-methylpropyl) - N4, N4-bis (phenylmethyl) -1H-imidazo [4,5-c] quinoline-2,4-diamine 1- {4-amino-2- [methyl (propyl) amino] -1H-imidazo [4,5 1- [4-amino-2- (propylamino) -1H-imidazo [4,5-c] quinolin-1-yl] -2-methyl -2-ol N4, N4-dibenzyl-1- (2-methoxy-2-methylpropyl) -N2-propyl-1H-imidazo [4,5-c] quinoline-2,4-diamine. • Saponins [Chapter 22 of Reference 133], which are a heterologous group of steroid glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. The saponin from the tree bark Molina 31

Quillaia saponaria, has been widely studied as an adjuvant. Saponin can also be obtained commercially from Smilax ornata (sarsaprilla), Gypsophilla paniculata (bridal veil) and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOM. QS21 is marketed as Stimulon ™. The saponin compositions were purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of producing QS21 is disclosed in reference 92. Saponin formulations may also comprise a sterol, such as cholesterol [93]. Combinations of saponins and cholesterols can be used to form unique particles called the immunostimulating complex (ISCOM) [chapter 23 of reference 133]. ISCOMs also typically include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA &amp; QHC. ISCOM are further described in references 93-95. Optionally, ISCOMS may be devoid of additional detergent [96]. A review of the development of saponin-based adjuvants can be found in references 97 &amp; 98. • Bacterial ADP ribosylating toxins (e.g., E.coli heat labile enterotoxin &quot; LT &quot;, cholera toxin &quot; CT &quot; or pertussis toxin &quot; PT &quot;) and derivatives thereof detoxified therefrom, such as mutant toxins, known as LT-K63 and LT-R72 [99]. The use of detoxified ADP ribosylating toxins as mucosal adjuvants is described in reference 100 and as parenteral adjuvants in reference 101. 32 Bioadhesives and mucoadhesives such as esterified hyaluronic acid microspheres [102] or chitosan and derivatives thereof [103]. Microparticles (e.g., a particle of ~ 100nm to ~ 150nm in diameter, more preferably ~ 200nm to ~ 30pm in diameter, or ~ 500nm to ~10m in diameter), formed from materials that are biodegradable and non-toxic ( for example a poly (α-hydroxy) acid, a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly (lactide-co-glycolide), being preferred, optionally being treated to have a negatively charged surface (e.g., with SDS) or a positively charged surface (for example, with a cationic detergent, such as CTAB). • The liposomes (Chapters 13 and 14 of reference 133). Examples of liposome formulations suitable for use as adjuvants are described in references 104-106. • Polyoxyethylene ethers and polyoxyethylene esters [107]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [108], as well as the polyoxyethylene alkyl ethers or ester surfactants, in combination with at least one additional nonionic surfactant, such as an octoxynol [109] . Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Muramyl peptides such as N-acetylmuramyl-L-threonyl-D-isoglutamine (&quot; thr-MDP &quot;), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylglucsaminyl N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy-propylamide (&quot; DTP-DPP &quot;, or &quot; Theramide ™), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- A protein preparation of the outer proteasome membrane prepared from a first Gram-negative bacterium, in combination with a compound of the general formula (I) with a liposaccharide (LPS) preparation derived from a second Gram-negative bacterium, wherein the outer membrane of proteosome protein and LPS preparations form a stable non-covalent adjuvant complex. Such complexes include &quot; IVX-908 &quot;, a complex composed of the outer membrane of Neisseria meningitidis and LPS and have been used as adjuvants for influenza vaccines. [110] • A poly mere polyoxidonium [111,112] or other oxidized N-polyethylene-piperazine derivative. • Methyl-5'-inosine-monophosphate (&quot; MIMF &quot;) [113]. A polyhydroxylated pyrrolizidine compound [114], such as one having the formula:

wherein R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, acyl, saturated or unsaturated, alkyl (e.g., cycloalkyl), alkenyl, alkynyl and aryl, or a pharmaceutically acceptable salt or a derivative thereof . Examples include, but are not limited to: casuarin, casuarina-6-Î ± -D-glucopyranose, 3-epi-casuarin, 7-epi-casuarin, 3,7-di-epi-casuarin, etc. • A CDld ligand, such as Î ± -glycosylceramide (eg α-galactosylceramide), phytosphingosine containing α-glycosylceramides, OCH, KRN7000 [(2S, 3S, 4R) -β- (β-galactopyranosyl) -2- (N-hexacosanoylamino) -1,3,4-octadecanetriol], CRONY-101, 3â € ²-O-sulfo-galactosylceramide, etc. • Inulin gamma [123] or a derivative thereof, such as algammulin. A compound of formula I, II or III, or a salt thereof:

1 II III

defined in reference 124, as 'ER 803058', 'ER 803732', 'ER 804053', 'ER 804058', 'ER 804059', 'ER 804442', 'ER 804680', 'ER 804764', 'ER 803022' or 'ER 804057', for example: 35

ER-803022:

or

Escherichia coli lipid A derivatives such as Î "M-174 (described in references 125 & 126). A formulation of a cationic lipid and a co-lipid (usually neutral) such as dimethylaminopropylpropanaminium myristoleiloxy bromide difihitanoylphosphatidyl ethanolamine (&quot; Vaxfectin &quot;) or aminopropyl-dimethyl-bis-dodecyloxypropanaminium bromide dioleoylphosphatidyl bromide -ethanolamine (&quot; GAP-DLRIE: DOPE &quot;). Formulations containing (±) -N- (3-aminopropyl) -N, N-dimethyl-2,3-bis (syn-9-tetradeceneyloxy) -1-propanaminium salts are preferred [127]. • Compounds containing lipids bound to a phosphate-containing acyclic skeleton, such as the TLR4 E5564 antagonist [128,129]:

These and other active adjuvants are discussed in more detail in references 133 and 134. The adjuvant (s) for use in the present invention may be Toll-like receptor (TLR) modulators and / or agonists. For example, they may be agonists of one or more of the human TLR1, TLR2, TLR3, TLR4, TLR7, TLR8 and / or TLR9 proteins. Preferred agents are the TLR7 (imidazoquinolines for example) and / or TLR9 agonists (for example, CpG oligonucleotides). These agents are useful for activating the innate immunity pathways.

A single vaccine may include two or more such adjuvants.

Antigens and adjuvants in a composition will typically be in the mixture.

Adjuvants of aluminum salts

Adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are conventional, but are used for convenience only, nor is it an accurate description of the actual chemical compound that is present (for example, see Chapter 9 of Reference 133). The invention may employ any of the &quot; hydroxide &quot; adjuvants &quot; or &quot; phosphate &quot;, which are commonly used as adjuvants.

Adjuvants known as &quot; aluminum hydroxide &quot; are typically oxyhydroxide salts of aluminum, which are usually at least partially crystalline. Aluminum oxyhydroxide, which may be represented by the general formula AIO (OH), can be distinguished from other aluminum compounds, such as aluminum hydroxide, AI (OH) 3, by infrared (IR) spectroscopy, in particular by presence of an adsorption band at 1070 cm -1 and a strong shoulder at 3090-3100 cm -1 [Chapter 9 of Reference 133]. The degree of crystallinity of an aluminum hydroxide adjuvant is reflected by the width of the half-height diffraction band (WHH), with poorly crystalline particles showing enlargement of the larger line due to smaller crystallite sizes. Surface area increases as WHH increases, and adjuvants with higher WHH values were seen as having a greater adsorption capacity for the antigen. A fibrous morphology (for example, as seen in the transmission electron microscope) is typical for aluminum hydroxide adjuvants. The amount of aluminum hydroxide adjuvants is typically about 11, i.e., the adjuvant itself has a surface charge positive at physiological pH. Adsorptive capacity between 1.8-2.6 mg protein per mg of Al +++ at pH 7.4 has been reported for aluminum hydroxide adjuvants. 38

Adjuvants known as &quot; aluminum phosphate &quot; are typically aluminum hydroxyphosphates, often also containing a small amount of sulfate (i.e., aluminum hydroxyphosphate sulfate). They can be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of the phosphate by the hydroxyl in the salt. Hydroxyphosphates generally have a PCy / Al molar ratio of between 0.3 and 1.2. The hydroxyphosphates can be distinguished from the strict AIPO4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm -1 (for example, when heated at 200 ° C) indicates the presence of structural hydroxyls (chapter 9 of ref. 133). The molar ratio of PO4 / AI3 &quot; 1 &quot; of an aluminum phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably of 0.95 ± 0.1. Aluminum phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminum hydroxyphosphate with PO4 / AI molar ratio of between 0.84 and 0.92, included in 0.6mg of Al3 + / ml. Aluminum phosphate will generally be in particles (for example of the plate type with the morphology observed in transmission electron micrographs). Typical particle diameters are in the range of 0.5-20 æm (e.g., about 5-10 æm) after any antigen adsorption. Adsorptive capacities between 0.7-1.5 mg of protein per mg of Al +++ at pH 7.4 have been reported for aluminum phosphate adjuvants. The zero phosphate (PZC) of aluminum phosphate is inversely related to the degree of phosphate substitution per hydroxyl, and this degree of substitution may vary depending on the reaction conditions and concentration of the reagents used to prepare the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in the solution (plus phosphate = PZC plus acid) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminum phosphates used according to the invention will generally have a PZC between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example about 5.7.

The aluminum salt suspensions used to prepare the compositions of the invention may contain a buffer (for example, a phosphate or histidine or Tris buffer), but this is not always necessary. The suspensions are preferably sterile and pyrogen-free. The suspension may include free aqueous phosphate ions, for example, present in a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM and most preferably about 10 mM. The suspensions may also comprise sodium chloride. The invention may employ a mixture of aluminum hydroxide and aluminum phosphate, as in DARONRIX ™. In this case, there may be more phosphate than aluminum hydroxide, for example a weight ratio of at least 2: 1, for example 5: 1 to 6: 1, 7: 1 to 8: 1, 9 : 1, etc. The concentration of Al ++ ++ in a composition for administration to a patient is preferably less than 10mg / ml &lt; for example 5 mg / ml, 4 mg / ml, &lt; 3 mg / ml, 2 mg / ml, &lt; 1 mg / ml, etc. A preferred range is between 0.3 and 1 mg / ml. A maximum of 0.85mg / dose is preferred.

The oil-in-water adjuvant emulsions 40

Oil-in-water emulsions have been found particularly suitable for use in the adjuvant of influenza virus vaccines. Various emulsions are known, and typically include at least one oil and at least one surfactant, the oil (s) and surfactant (s) being biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion are generally less than 5 μm in diameter, and may even have a sub-micron diameter, with these small dimensions to be achieved with a microfluidizer to provide stable emulsions. Drops having a size of less than 220 nm are preferable since they can be subjected to sterilization filtration. The invention may be used with oils, such as those from an animal (such as fish) or from vegetable origin. Sources of vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil and olive oil, the most commonly available, exemplify nut oils. Jojoba oil may be used, for example obtained from jojoba beans. The seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the group of cereals, corn oil is the most readily available, but other cereal grain oil such as wheat, oats, rye, rice, teff, triticale and the like can also be used. 6-10 fatty acid esters of glycerol and 1,2-propanediol, although not naturally occurring in seed oils, may be prepared by hydrolysis, separation and esterification of suitable materials from the nuts and seeds oils. Fats and oils from mammalian milk are metabolizable and can therefore be used in the practice of this invention. Procedures for the separation, purification, saponification and other means necessary to obtain pure oils of animal origin are well known in the art. Many fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oil and whale oil as spermaceti exemplify various fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred here. Squalane, the saturated analogue of squalene, is also a preferred oil. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known in the art. Other preferred oils are tocopherols (see below). Mixtures of oils may be used.

Surfactants can be classified by their &quot; HLB &quot; (hydrophile / lipophilic balance). Preferred surfactants of the invention have an HLB of at least 10, preferably at least 15, and most preferably at least 16. The invention may be used with surfactants including, but not limited to: polyoxyethylene sorbitan esters surfactants (commonly referred to as such as Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trade name DOWFAX ™, such as block EO / PO linear copolymers; octoxynols, which may vary in the number of repeats of the ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin), nonylphenol ethoxylates, such as the Tergitol ™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30) and sorbitan esters (commonly known as SPANs) such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Preferred surfactants for inclusion in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.

Mixtures of surfactants may be used, for example mixtures of Tween 80 / Span 85. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol, such as t-octylphenoxypolyethoxyethanol (Triton X -100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

Preferred amounts of surfactants (wt%) are as follows: 0.01 to 1% polyoxyethylene sorbitan esters (such as Tween 80), in particular about 0.1%; 0.001 to 0.1% of octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents of the Triton series), in particular 0.005-0.02%; 0.1 to 20% polyoxyethylene ethers (such as laureth 9), preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific water-in-oil adjuvant emulsions useful with the invention include, but are not limited to: • A submicron squalene emulsion, Tween 80 and Span 85. The emulsion composition by volume may be about 5% squalene , about 0.5% polysorbate 80 and about 0.5% Span 85. By weight these proportions are 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant is known as 'MF59' [130-132], as described in more detail in Chapter 10 of Reference 133 and Chapter 12 of Reference 134. The MF59 emulsion advantageously includes citrate ions, e.g. mM sodium citrate buffer. • An emulsion of squalene, a tocopherol and Tween 80. The emulsion may include phosphate buffered saline. It may also include Span 85 (for example, 1%) and / or lecithin. These emulsions may have from 2 to 10% squalene, 2 to 10% tocopherol and 0.3-3% Tween 80, and the weight ratio of squalene: tocopherol is preferably 1 (e.g., 90), as this provides a more stable emulsion. Squalene and Tween 80 may be present in the volume ratio of about 5: 2, or in a weight ratio of about 11: 5. Such an emulsion may be effected by dissolving Tween 80 in PBS to give a 2% solution followed by mixing 90 ml of this solution with a mixture of (5 g DL-α-tocopherol and 5 ml squalene), then microfluidization of the mixture. The resulting emulsion may have submicron oil droplets, for example, with an average diameter of between 100 and 250 nm, preferably about 180 nm. • An emulsion of squalene, a tocopherol, and a Triton detergent (for example, Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer. An emulsion comprising a polysorbate (e.g., polysorbate 80), with a Triton detergent (e.g.

Triton X-100) and a tocopherol (for example an a-tocopherol succinate). The emulsion may comprise the three components in a mass ratio of about 75:11:10 (for example, 750pg / ml polysorbate 80, 110pg / ml Triton X-100 and 100pg / ml α-tocopherol succinate), and the The concentrations used should include any contribution of these antigen components. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer. • An emulsion of squalane, polysorbate 80 and poloxamer 401 (&quot; Pluronic ™ L121 &quot;). The emulsion may be formulated in phosphate buffered saline, pH 7.4. This emulsion is a carrier useful for delivery of muramyl dipeptides, and has been used with treonyl-MDP in the &quot; SAF-1 &quot; adjuvant &quot; [135] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without Thr-MDP, as in the &quot; AF &quot; [136] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidization is preferred. An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether of hydrophilic nonionic surfactant (for example, polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (for example, a sorbitan ester or a mannide ester , such as sorbitan monoleate or 'Span 80'). The emulsion is preferably heat-reversible and / or has at least 90% of the oil (by volume) droplets having a size of less than 200 nm, [137]. The emulsion may also include one or more of the following: alditol; a cryoprotective agent (for example, a sugar, such as dodecylmaltoside and / or sucrose) and / or an alkylpolyglycoside. Such emulsions may be lyophilized. An emulsion with 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a nonionic surfactant. As described in reference 138, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. The submicron droplet sizes are advantageous. • A water-in-oil submicron emulsion of a non-metabolizable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophilic conjugate (such as GPI-0100, described in reference 139, produced by addition of an aliphatic amine to desacylsaponine via the carboxyl group of glucuronic acid), bromide of dimethyldoctadecylammonium and / or N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine. An emulsion in which a saponin (e.g., QuilA or QS21) and a sterol (e.g., a cholesterol) are associated as helical micelles [140]. An emulsion comprises a mineral oil, a lipophilic ethoxylated nonionic fatty alcohol, and a nonionic hydrophilic surfactant (for example, an ethoxylated fatty alcohol and / or a polyoxyethylene-polyoxypropylene block copolymer) [141]. An emulsion comprises a mineral oil, a nonionic ethoxylated hydrophilic fatty alcohol and a nonionic lipophilic surfactant (for example, an ethoxylated fatty alcohol and / or a polyoxyethylene-polyoxypropylene block copolymer) [141]. 46

Preferred water-in-oil emulsions of the invention comprise squalene.

The emulsions may be mixed with the antigen extemporaneously at the time of delivery. Thus, the adjuvant and the antigen may be kept separately in a packaged or distributed vaccine, ready for the final formulation, at the time of use. The antigen will generally be in an aqueous form such that the vaccine is finally prepared by mixing two liquids. The ratio of the two liquids per volume of mixture may vary (for example, 5: 1-1: 05), but generally is about 1: 1. If the antigen and the emulsion are stored separately in the multidose kit, then the product may be presented as a vial containing 2.5 ml of emulsion and a vial containing 2.5 ml of aqueous antigen, by admixture, to give 5 ml vaccine with adjuvant, for example 10 x 0.5 ml per dose.

After the antigen and the adjuvant are mixed, the hemagglutinin antigen generally remains in aqueous solution, but may be distributed around the oil / water interface. In general, little or no hemagglutinin will enter the oil phase of the emulsion.

When a composition includes a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols may be used, but α-tocopherols are preferred. Tocopherol may take several forms, for example different salts and / or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-α-tocopherol and DL-α-tocopherol 47 may both be used. Tocopherols are advantageously included in vaccines for use in elderly patients (eg, 60 years or older) because vitamin E has been reported to have a positive effect on the immune response in this group of patients [142]. They also have antioxidant properties that can help stabilize emulsions [143]. A preferred α-tocopherol is DL-Î ± -tocopherol, and the preferred salt of this tocopherol is succinate. The succinate salt was found to cooperate with TNF-related ligands in vivo. In addition, α-tocopherol succinate is known to be compatible with influenza vaccines and a preservative to be useful as an alternative to mercury compounds [13].

Oligonucleotides Immunostimulators

Immunostimulatory oligonucleotides may include nucleotide modifications / analogues such as osteothioate modifications and may be double-stranded or (except for single-stranded RNA). References 144, 145 and 146 disclose the possible analogous substitutions, for example the substitution of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is discussed further in references 147-152. A CpG sequence may be directed to TLR9, such as the GTCGTT or TTCGTT motif [153]. The CpG sequence may be specific for the induction of a Th1 immune response, such as a CpG-A ODN (oligodeoxynucleotide), or may be more specific for the induction of a B cell response, such as a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in references 154-156.

Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed such that the 5 'end is already accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3 'ends to form &quot; immunomers &quot;. See, for example, references 153 and 157-159. A useful CpG adjuvant is CpG7909, also known as ProMune ™ (Coley Pharmaceutical Group, Inc.).

As an alternative, or additionally, to the use of CpG sequences, the TPG sequences [160] may be used. These oligonucleotides may be free of unmethylated CpG motifs. The immunostimulatory oligonucleotide may be pyrimidine rich. For example, it may comprise more than one consecutive thymidine nucleotide (e.g., TTTT, as described in ref. 160), and / or may have a nucleotide composition with> 25% thymidine (e.g.> 35%, > 40%,> 50%,> 60%,> 80%, etc.). For example, it may comprise more than one consecutive cytosine nucleotide (e.g., CCCC as described in ref. 160), and / or may have a nucleotide composition with &gt; 25% cytosine (e.g. &gt; 35%, &gt; 40%, &gt; 50%, &gt; 60%, &gt; 80%, etc.). These oligonucleotides may be free of unmethylated CpG motifs.

Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise less than 100 nucleotides.

An adjuvant particularly based on immunostimulatory oligonucleotides is known as IC31 ™ [161]. Thus, an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e.g., between nucleotides 15-40), including at least one (and preferably multiple) CPI motifs, and (ii) a polymer polypeptide, such as an oligopeptide (e.g., between 5-20 amino acids), including at least one (and preferably multiple) tripeptide sequence Lys-Arg-Lys (s). The oligonucleotide may be a deoxynucleotide comprising the sequence 26-mer 5 '(IC) 13-3' (SEQ ID NO: 14). The polycationic polymer may be a peptide comprising the 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 15). Lipid A 3-de-O-acylated monophosphoryl 3-O-acylated 3dMPL (also known as 3-de-O-acylated monophosphoryl lipid A or 3-O-desacyl-4'-monophosphoryl lipid A is an adjuvant in which the 3-position of the reducing end of glucosamine The monophosphoryl was de-acylated. 3dMPL was prepared from a non-heptose mutant of Salmonella minnesota, and is chemically similar to lipid A, but does not have an acid labile phosphoryl group and a base-labile acyl group. activates the monocyte / macrophage cells and stimulates the release of various cytokines, including IL-1, IL-12, TNF-α and GM-CSF (see also reference 162). The preparation of 3dMPL was originally described in reference 163 (E.g., having 3, 4, 5 or 6 acyl chains, which may be of different lengths) .The two monosaccharides of glucosamines (also known as glycoprotein monosaccharides) may be in the form of a mixture of related molecules which vary by their acylation. such as 2-deoxy-2-amino-glucose ) are N-acylated on their carbons in the 2-position (i.e., in the 2 and 2 'positions), and there is also O-acylation at the 3' position. The group attached to carbon 2 has the formula -NH-CO-CH 2 -CR 1 R 1 '. The group attached to the 2 'carbon has the formula -NH-CO-CH 2 -CR 2 R 2'. The group attached to the 3 'carbon has the formula -O-CO-CH 2 CR 3 R 3'. The representative structure is: 50

The groups R 1, R 2 and R 3 are each independently - (CH 2) n -CH 3. The value of n is preferably 8 to 16, more preferably 9 to 12, and is most preferably 10.

The R1 ', R2' and R3 'groups may each be independently: (a) -H; (b) -OH, or (c) -O-CO-R4, wherein R4 is -H or -CH2) m -CH3, wherein the value of m is preferably 8 to 16, and is most preferably 10 , 12 or 14. In the 2-position, m is preferably 14. In the 2-position m is preferably 10. In the 3 'position m is preferably 12. The groups R1', R2 'and R3 are therefore preferably dodecanoic acid-O-acyl groups, tetradecanoic acid or hexadecanoic acid.

When all R1 ', R2' and R3 'are -H, then 3dMPL has only 3 acyl chains (one at each of the 2, 2' and 3 'positions). When only two of R1, R2 and R3 are -H, then 3dMPL may have 4 acyl chains. When only one of R1, R2 'and R3' is -H, then 3dMPL may have 5 acyl chains. When none of R1 ', R2' and R3 'are -H, then 3dMPL may have 6 acyl chains. The adjuvant 3dMPL used according to the invention may be a mixture of such forms having from 3 to 6 acyl chains, but it is preferable to include 3dMPL with acyl chains in the mixture, and in particular to ensure that the hexaacyl chain form does , at least 10% by weight of the total of 3dMPL h for example 20%, 30% h, 40%, 50% or more. 3dMPL with 6 acyl chains was found to be the most active form of adjuvant.

Thus, the most preferred form of 3dMPL for inclusion in the compositions of the invention is as follows:

Where 3dMPL is used in the form of a mixture, wherein references to amounts or concentrations of 3dMPL in compositions of the invention refer to the combined 3dMPL species in the blend.

Under aqueous conditions, 3dMPL may form micellar aggregates or particles of different sizes, for example with a diameter &lt; 150 nm or &gt; 500 nm. Either or both may be used with the invention, and the best particles may be selected by routine testing. Smaller particles (for example, small enough to give a clear aqueous suspension of 3dMPL) are preferred for use according to the invention, because of their high activity [164]. Preferred particles have a mean diameter of less than 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and may even have an average diameter of less than 100 nm. In most cases, however, the average diameter will not be less than 50nm. Such particles are small enough to be suitable for sterilization by filtration. The particle diameter can be evaluated by the routine technique of dynamic light scattering, which reveals an average particle diameter. When a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this significant, but at least 50% by number (e.g., A 60%, A 70%, h 80%, &gt; 90%, or more) of the particles will have a diameter within the range x ± 25%. The 3DMPL can be advantageously used in combination with an oil-in-water emulsion. Substantially all of the 3dMPL may be located in the aqueous phase of the emulsion. 3DMPL can be used alone, or in combination with one or more additional compounds. For example, the use of 3dMPL, in combination with QS21 [165] saponin (including, in an oil-in-water emulsion [166]), with an immunostimulatory oligonucleotide, both with QS21 and an immunostimulatory oligonucleotide, is known as with aluminum phosphate [167], with aluminum hydroxide [168], or with both aluminum phosphate and aluminum hydroxide.

Pharmaceutical Compositions

The compositions of the invention are pharmaceutically acceptable and are typically in the aqueous form. They may include components other than the antigen (and, if appropriate, the adjuvant), for example, which typically include one or more pharmaceutical carrier (s) and / or excipient (s). An in-depth discussion of the components is available from ref. 169. The composition may include preservatives such as thiomersal (e.g. at 10pg / ml) or 2-phenoxyethanol. It is preferred, however, that the vaccine should be substantially free of (i.e., less than 5pg / ml) mercury material, for example thiomersal free [13,170]. Vaccines containing no mercury are most preferred. Vaccines free of preservatives are particularly preferred.

To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present in the range of 1 to 20 mg / ml. Other salts which may be present include potassium chloride, potassium phosphate dibasic, disodium phosphate dehydrated, magnesium chloride, calcium chloride, etc.

The compositions will generally have an osmolarity between 200 mOsm / kg and 400 mOsm / kg, preferably between 240-360 mOsm / kg, and more preferably will fall within the range of 290-310 mOsm / kg. Osmolarity has been previously reported 54 as not having an impact on pain caused by vaccination [171], but maintaining osmolality in this range, however, is preferred.

The compositions may include one or more buffers. Typical buffers include: phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (in particular with an aluminum hydroxide adjuvant); or citrate buffer. Buffers will typically be in the range of 5-20 mM. The pH of the composition generally ranges from 5.0 to 8.1, and more typically from 6.0 to 8.0, for example from 6.5 to 7.5, from 7.0 to 7.8. A process may therefore include a step of adjusting the pH of the vaccine prior to bulk packaging. The composition is preferably sterile. The composition is preferably, for example, non-pyrogenic, containing &lt; 1 EU (endotoxin unit, a standard measure) per dose, and preferably &lt; 0.1 EU per dose. The composition is preferably gluten-free. The composition may include material for a single immunization, or may include material for multiple immunizations (i.e. a &quot; multidose &quot; kit e.g. for 10 doses). Inclusion of a preservative is preferred on a multidose basis. As an alternative (or in addition) to include a preservative in multidose compositions, the compositions may be contained in a container having an aseptic filler adapter for removal of material. 55

Influenza vaccines are typically administered in a dosage volume of about 0.5 ml, although a half dose (i.e., about 0.25 ml) may be administered, for example, to children (e.g. up to 36 months of age).

The compositions and kits are preferably stored at 2 ° C to 8 ° C. They should not be frozen. They should ideally be kept out of direct light.

Kits

The kits may contain more than one composition of the invention, for example, an initiation composition and a strengthening composition. The two components of the kit will be maintained separately as they are administered to a patient at substantially different times.

Each individual vaccine in a kit can be prepared for use, or may be ready for extemporaneous preparation at the time of delivery. This extemporaneous arrangement allows the adjuvant and the antigen to be maintained separately until the time of use, which is particularly useful when using a water-in-oil emulsion adjuvant.

When a vaccine is prepared extemporaneously, its components are physically separated from one another within the kit, and this separation can be achieved in a number of ways. For example, the two components may be in two separate containers, such as vials, for example an antigen vial and an emulsion vial. The contents of the two vials may then be mixed, for example, by withdrawing the contents of one vial and adding it to the other vial, or by separately removing the contents of both vials and their mixture in a third vial. In a preferred arrangement, one of the components of the kit is a syringe and the other is a container such as a vial. The syringe may be used (for example, with a needle) to insert its contents into the second mixing vessel, and the mixture can then be withdrawn into the syringe. The mixed contents of the syringe may then be administered to a patient, typically by means of a new sterile needle. The packaging of a component in a syringe eliminates the need to use a separate syringe for administration to the patient.

In another preferred arrangement, the two components of a vaccine are held together, but separated, in the same syringe, for example a double-chamber syringe, such as those described in references 172-179 etc. When the syringe is activated (for example, during administration to a patient) then the contents of the two chambers are mixed. This arrangement avoids the need for a separate blending step at the time of use.

When a vaccine is prepared extemporaneously, its components will generally be in aqueous form. In some embodiments, one component (typically the antigen component rather than the adjuvant component) is in the dried form (e.g., in a lyophilized form), the other component being in the aqueous form. The two components may be mixed in order to reactivate the dry component and give an aqueous composition for administration to a patient. A lyophilized component will typically be located within a vial rather than the syringe. Dry components may include stabilizers, such as lactose, sucrose or mannitol, as well as mixtures thereof, for example, lactose / sucrose mixtures, sucrose / mannitol blends, etc. One possible arrangement uses an aqueous adjunct component in a pre-filled syringe, and a lyophilized antigen component in a vial.

Packing of kit components or components

Containers suitable for the compositions of the invention (or kit components) include vials, syringes (for example, disposable syringes), nasal sprays, etc. These containers should be sterile.

Where a composition / component is located in a vial, the vial is preferably made of glass or plastic material. The vial is preferably sterilized before the composition is added. To avoid problems with latex-sensitive patients, the vials are preferably sealed with a latex-free cork, and the absence of latex is preferred throughout the packaging material. The vial may include a single dose of vaccine, or may include more than one dose (a multidose vial), for example, 10 doses. Preferred vials are made of colorless glass. The vial may have a cap (e.g., a Luer cone) adapted such that a pre-filled syringe may be inserted into the cap, the contents of the syringe may be expelled into the vial (for example to reconstitute the lyophilized material), and the contents of the vial can be removed back into the syringe. Upon removal of the syringe from the vial, a needle may then be attached and the composition may be administered to a patient. The cap is preferably located within a seal or cover such that the seal or cover has to be removed before the cover can be accessed. A vial may have a closure which allows the aseptic removal of its contents, particularly for multi-dose vials.

Whenever a composition / component is packaged inside a syringe, the syringe may have a needle attached thereto. If a needle is not attached, a separate needle may be provided with the syringe for assembly and use. This needle can be coated. Safety needles are preferred. 23 inch, 1 inch 25 gauge and 5/8 inch 25 gauge needles are typical. Syringes may be provided with detachable labels in which lot number, flu season, and expiration date of the contents may be printed to facilitate record keeping. The plunger of the syringe preferably has a stop to prevent the plunger from being accidentally removed during aspiration. The syringes may have a rubber cap and / or plunger. Disposable syringes contain a single dose of vaccine. The syringe generally has a tip cap for sealing the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are then packaged separately the needle is preferably equipped with a butyl rubber retainer. Preferred syringes are those marketed under the trade name &quot; Tip-Lok &quot; ™. Butyl rubber is also a suitable material for the stoppers of multi-dose vials, for example in kits.

The containers may be labeled to show a half-dose volume, for example, to facilitate administration to 59 children. For example, a syringe containing a dose of 0.5 ml may have a mark showing a volume of 0.25 ml.

If a glass container (for example, a syringe or a vial) is used, then it is preferable to use a container made of a borosilicate glass rather than a glass of soda lime.

A kit or composition may be packaged (eg in the same box) with a leaflet including details of the vaccines for example, instructions for administration, information on the antigens included in the vaccine, etc. The instructions may also contain warnings, for example, to maintain an adrenaline solution readily available in case of anaphylactic reaction after vaccination, etc.

Administration of the vaccine

The compositions of the invention are suitable for administration to human patients. The raised immune response according to the invention generally includes an antibody response, preferably a protective antibody response. Methods for evaluating antibody responses, the ability to neutralize and protect after virus vaccination are well known in the art. Studies in humans have shown that the titers of hemagglutinin antibodies to the human influenza virus are correlated with protection (from a serum titre of hemagglutination inhibition serum of about 30-40 to about 50% provides protection against infection by a homologous virus) [180]. Antibody responses are typically measured by inhibition of haemagglutination, by microneutralization, by simple radial immunodiffusion (SRID) and / or by simple radial haemolysis (SRH). These assay techniques are well known in the art.

The compositions of the invention may be administered in a variety of ways. The most preferred immunization route is by intramuscular injection (for example, the arm or leg), but other available routes include subcutaneous, intranasal [181-183], oral [184], intradermal [185,186], transcutaneous, transdermal injection [187], etc.

The vaccines of the invention can be used both to treat both children and adults. Influenza vaccines are currently recommended for use in pediatric and adult immunization, from the age of 6 months. Thus, the patient may be less than 1 year of age, 1-5 years, 5-15 years, 15-55 years, or at least 55 years of age. Patients preferred to receive the vaccines are the elderly (for example, at age 50, at age 60, preferably at age 65), young people (eg 5 years), hospitalized patients, health workers, armed and military service, pregnant women, the chronically ill, the immunocompromised, patients taking an antiviral compound (for example, an oseltamivir compound or zanamivir, see below) within 7 days of receiving the vaccine, people with egg allergies, and persons traveling abroad. However vaccines are not only suitable for these groups and may be used more generally in the population. For pandemic strains, administration to all age groups is preferred. 61

Preferred compositions of the invention satisfy 1, 2 or 3 of the criteria for CPMP efficacy. In adults (18-60 years), these criteria are: (1) 70% seroprotection, (2) 40% seroconversion and / or (3) a GMT increase of 2.5 times. In the elderly (&gt; 60 years), these criteria are: (1) &gt; 60% seroprotection, (2) &gt; 30% seroconversion and / or (3) a GMT increase of A 2-fold. These criteria are based on open studies with at least 50 patients.

In embodiments of the initiation-booster invention, a patient is subjected to a multiple dose schedule. In a multiple dose schedule the various doses may be administered by the same or different routes, for example, a parenteral initiation and a mucosal boost, a mucosal initiation and a parenteral enhancement, etc. Multiple doses will typically be administered at least 1 week apart (for example, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks , about 16 weeks, etc.).

Where the invention involves immunizing (booster) a patient who has been previously immunized against a different H5 clade, the booster dose may be administered several months after the previous dose, for example at least 6 months, 9 months, 12 months, 18 months, 24 months, 36 months, 48 months, 60 months or more.

In embodiments wherein the composition includes an HA of more than one influenza A H5 virus clade, this composition may be administered as a single dose or multiple dose. Administration of more than one dose (generally two doses) is particularly useful in immunologically weak patients 62 for example, for people who have never received an influenza vaccine before, or for vaccination against a new subtype of HA such as H5. As above, multiple doses will typically be administered at least 1 week apart (for example, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about eight weeks, about 10 weeks, about 12 weeks weeks, approximately 16 weeks, etc.).

The compositions of the invention may be administered to patients substantially at the same time as (for example, during the same medical visit or visit to a healthcare provider or vaccination center) with other vaccines, for example substantially at the same time as measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a chickenpox vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a b-type influenza vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a conjugated meningococcal vaccine (such as a tetravalent AC-W135-Y vaccine), a respiratory syncytial virus vaccine, a vaccine pneumococcal conjugate, etc. Administration substantially at the same time as a pneumococcal vaccine or a meningococcal vaccine is particularly useful in elderly patients.

Likewise, the compositions of the invention may be administered to patients substantially at the same time as (e.g., during the same medical visit or visit with a healthcare provider) an antiviral compound, and in particular an antiviral active compound against the virus of influenza A (for example oseltamivir and / or zanamivir). Such antivirals include neuraminidase inhibitors, for example a (3R, 4R, 5S) -4-acetylamino-5-amino-3- (1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid or 5- ( acylamino) -4 - [(aminonoiminoethyl) amino] 2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-e-enonic acid, including esters (for example, ethyl esters) and their salts (for example, the phosphate salts). A preferred antiviral is (3R, 4R, SS) -4-acetylamino-5-amino-3- (1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid, ethyl ester, phosphate (1: 1), also known as seltamivir phosphate (Tamiflu ™).

General The term &quot; comprising &quot; includes &quot; including &quot; as well as &quot; consisting &quot; for example, a composition &quot; comprising &quot; X may consist exclusively of X or may include something additional, for example X + Y. The word &quot; substantially &quot; does not delete &quot; completely &quot; for example, a composition which is &quot; substantially free &quot; Y may be completely Y-free. Whenever necessary, the word &quot; substantially &quot; may be omitted from the definition of the invention. The term &quot; about &quot; with respect to a numerical value of x means, for example, x ± 10%. Unless otherwise specified, a process comprising a step of mixing two or more components requires no specific order of the blend. Thus, the components can be mixed in any order. When there are three components then two components can be combined with one another, and then the combination can be combined with the third component, etc. 64

Where animal (and particularly bovine) materials are used in cell culture, they must be obtained from sources that are free of transmissible spongiform encephalopathies (TSEs), and in particular bovine spongiform encephalopathy (BSE) free. In general, cell culture is preferred in the absence of total animal derived materials.

When a compound is administered to the body as part of a composition, then that compound may alternatively be replaced with a suitable prodrug.

When a cell substrate is used for rearrangement or reverse genetics procedures, it is preferably one that has been approved for use in the production of human vaccine, for example, in Ph Eur general chapter 5.2.3.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 and 2 show the phylogenetic trees of H5 strains, made from references 3 and 189.

MODES FOR CARRYING OUT THE INVENTION Human study I

Patients were immunized with a vaccine prepared from the H5N3 A / duck / Singapore / 1997 (clade 0) strain. The vaccine was without adjuvant (group 2) or was adjuvanted with the MF59 oil-in-water emulsion (group 1). A third group of patients (group 3) did not receive the H5N3 vaccine.

In a later influenza season (after at least 6 years later), patients were immunized with a flu vaccine prepared from a H5N1 strain (A / Vietnam / 1194/2004 clade = 1; clade 2 also can be used). Two doses were given on days 0 and 21, both containing 7.5pg of hemagglutinin and adjuvant MF59. Pre- and post-vaccination antibodies to antigenically different H5 viruses were measured by inhibition of haemagglutination (HAI), neutralizing antibody (MN) and single radial haemolysis (HRS).

The results are presented in Table I. Briefly, patients who had previously been immunized by the clade 0 vaccine showed a faster and better immune response against the new clade than non-primary patients. Within the primary group, patients who received an initial dose of adjuvant showed a better immune response than patients who received an initial dose without adjuvant. The geometric mean of antibody titres and sero-responses were significantly higher in vaccinated individuals than in non-immunized individuals. At day 7, after a dose of vaccine, 80% of the patients in group 1 had reached the HAI seroprotective titers of 1:40 h for all clades 1, 2.1, 2.2, 2.3 and the variants of the H5 avian virus tested, as well as with the original antigen of clade 0 A / Duck / Singapore / 97. In addition, there was no evidence to suggest that initially vaccinated individuals have responded to their original original antigen 66, alleviating concerns about &quot; original antigenic sin &quot; [190].

Thus, it is possible to rapidly induce protective antibody responses against various H5N1 influenza viruses following a dose of vaccine in subjects previously prepared several years earlier with the vaccine prepared from a strain of a different antigenically and genetically distant H5 clade.

Further details of this clinical study are available from reference 191. As noted therein, the geometric mean titers of antibodies against a clade I strain or a clade strain 2.2 were significantly higher among the vaccinated individuals (groups 1 and 2) than between subjects not immunized (group 0). From day 14 onwards, antibody titers for both viruses were significantly higher in the adjuvant initial group (group 1) than in the baseline group without adjuvant (group 2). The highest titers were observed on day 14 in group 1. There was no relationship between the post-vaccination titre and the number of previous doses of the H5N3 vaccine or its antigen content. At day 7, at least 80% of patients in group 1 had titers of at least 1:40 for all wild-type viruses tested in the haemagglutination inhibition assay.

Pandemic propagation modeling shows that maximum viral transmission reduction is achieved by inducing a response within 2 weeks after the outbreak of the pandemic. Because the two doses of the vaccine would be needed, rapid implantation of the vaccine would thus be difficult. This human study, however, indicates that subjects with initiation of the H5 antigen (in particular the adjuvanted H5 antigen) induce a long-term, rapidly mobilized immune memory following the administration of an antigenically distinct, low-dose vaccine (other than H5 clade HA).

In subsequent studies, cross-reactive memory B cells for clade 1 H5N1A / Vietnam / 1194/2004 were detected at comparable frequencies in the blood of all three groups of patients at the baseline. However, three weeks after the first booster dose, patients in group 1 had a significantly higher number of H5N1 virus-specific memory B cells than groups 2 and 3, suggesting that initiation with the H5N3 vaccine with adjuvant had induced an amount of memory B cells with greater cross-reactivity to H5N1. Consistently, patients in group 1 had faster and greater antibody responses than patients in groups 2 and 3, and patients in groups 1 and 2 responded significantly better and faster than patients in group 3. day after a dose of clade 1 vaccine, all patients in group 1 had achieved seroconversion to various antigenically distinct wild type pathogenic viruses of clades 0, 1, 2.1.3, 2.2 and 2.3.4, while in group 2 the comparable seroconversion rates were observed only on day 14. On the other hand, two doses of a clade 1 vaccine were required for group 3 patients to achieve 80% seroconversion rates for clade 0 and clade 1 viruses.

Human study II 68

Adult and elderly patients received two doses of initiation (day 0 and day 21) of an H5N1 vaccine with MF59 adjuvant. The virus strain was A / Vietnam / 1194/2004, which is classified in clade 1. Immune responses were assessed on day 43 and all three CPMP criteria were met: seroprotection and seroconversion were at least 80%, and the GMT increase was at least five times.

About 18 months after the two initiation doses, up to 60 patients received an additional dose of H5N1 vaccine with MF59 adjuvant, but based on strain A / turkey / Turkey / 1/05, which is classified in clade 2. Samples of serum are collected immediately before this dose and then at 7 and 21 days later. Immunogenicity is assessed by HI, SRH and MN in serum samples.

Human trials III and IV The NCT00703053 trial uses a clade 1 (A / Vietnam / 1203/04) vaccine and / or a clade 2 vaccine (A / Indonesia / 05/05). Adults without prior exposure to the H5 strains received: (a) a clade 1 vaccine at day 0 and a clade 2 vaccine at day 28; (b) a clade 1 vaccine at day 0 and a clade 2 vaccine at day 180; or (c) the combination of clade I and clade 2 vaccines on days 0 and 28. Suitable controls are also included, receiving only one clade 1 vaccine or one clade 2 vaccine, but not both. The total antigen dose of each time is 90pg HA, which is either 90pg from a single strain for groups (a) and (b), or from 2x45pg of each strain for group (c). The study uses inactivated subvibrand vaccines without adjuvant. The trial will not be completed before 2010. 69 The NCT00680069 trial involves administering a single dose of a clade 2 vaccine (A / Indonesia / 05/05) to patients who have previously received a clade 1 (A / Vietnam / 1203/04). The dose of antigen is 15pg or 90pg of an inactivated subvirus vaccine. The study will not be completed before 2009.

Studies in Rats I and II (DNA immunization; for reference)

In independent experiments [192], adenovirus vectors expressing HA from virus A / Vietnam / 1203/04 (clade 1) and A / Indonesia / 05/05 (clade 2) were constructed.

Vectors were used either separately or in combination to immunize BALB / c mice. The total dose was 108 pfu of vectors, with the group receiving a combination of 5x107 pfu of each vector. Four weeks later the rats received a booster containing the same vaccine construct (s) and blood was obtained 3 weeks later for the detection of neutralizing antibodies and antibodies that inhibit haemagglutination.

Rats vaccinated with any of the vectors alone produced antibodies that inhibit haemagglutination and neutralization, but no cross-reactivity was detected. However, rats vaccinated with both vectors elicited neutralizing antibody titers to protect against viruses from both clades.

In a similar study [193] the mice received a combination of five or ten hemagglutinin DNA immunogens as 70 vectors of the expression plasmid. The first combination of five HAs was from strains in clades 0, 2.2 (three strains) and 2.5. The second combination of five HAs was from strains in clades 0, 1, 2.1.3, 2.2 and 2.3.4. Valence 10 was a combination of these 10 strains. The vaccines induced antibodies that neutralized the various strains of HPAI H5N1.

Chickens were also tested using valence DNA vaccines of this type [193]. The strains were A / Vietnam / 1203/04 (clade 1), A / Anhui / 1/05 (clade 2.3.4) and A / Indonesia / 05/05 (clade 2.1.3). The chickens were protected against the disease.

Study in rats III

As described in detail in reference 8, the mice received a bivalent vaccine including hemagglutinin H5 from strains A / Vietnam / 1203/2004 (clade 1) or A / Indonesia / 05/2005 (clone 2). Two types of bivalent vaccine were tested: one based on recombinant H5 hemagglutinin and one based on VLPs. No vaccine has included an extrinsic adjuvant, but, compared to recombinant proteins, VLP may provide an intrinsic adjuvant effect because of its nature of particles. Each monovalent VLP was used as a control. Antigens were expressed in Sf9 insect cells from bacmids. The antigens in the bivalent vaccines were mixed at a weight ratio of HA of 1: 1.

Immune responses were assessed by quantitative ELISA and HAI. All mice vaccinated with the bivalent VLP mixture induced HAI antibodies against both VietNam / 1203/2004 virus (GMT 115 ± 36) and virus 71

Indonesia / 05/2005 (80 ± 0). In contrast, only 33% of the mice vaccinated with a recombinant protein blend without adjuvant had a HAI titre against Indonesia / 05/2005 virus (36 ± 12).

Immune responses were also evaluated in a lethal challenge study. The challenge strains were PR8 / 34 reformulated from the two vaccine strains. Unvaccinated mice that were challenged with the virus lost &gt; 20% of the original weight at day 6 post-infection. Mice vaccinated with the bivalent mixture of recombinant HA proteins lost -15% of their original body weight when challenged by either the challenge strain of clade I or clade 2. Rats vaccinated with clade VLPs 1, clade 2 VLPs , or VLP mixture and challenged with virus clade 1 had no weight loss and no clinical signs of infection. Rats vaccinated with clade 1 VLPs and challenged with clade 2 virus were not protected from challenge, dying by day 6 post challenge. Mice vaccinated with clade 2 VLP or the VLP blend were all protected from challenge with clade 2 virus.

Thus, VLPs from clades 1 and 2 were both immunogenic in rats and protected against challenge with the homologous strain virus. Immunogenicity was retained if a bivalent mixture of VLPs was used. In addition, rats that received the divalent VLPs were protected against challenge by clade 1 or clade 2 viruses. As reported in reference 8: &quot; These results are highly significant and demonstrate that a multivalent vaccine against H5N1 appears to be a a plausible strategy to combat the diversity of H5N1 influenza clades and subclasses. &quot; 72

It will be understood that the invention has been described by way of examples only and modifications may be made while remaining within the scope of the invention. 73

Table 1

Measure G 1 G 2 G 3% of patients with the titer H140 on day 0 0 3 on day 8 75 56 12 on day 15 90 60 9 on day 22 75 58 13 on day 43 75 58 45% of patients with seroconversion on day 8 75 56 8 on day 15 90 60 10 on day 22 75 58 14 on day 43 75 58 45 Geometric means of HI on day 1 4 4 4.9 on day 8 72 51 5.9 on day 15 256 79 7.3 on day 22 112 52 Co on day 43 95 44 26 Relative ratio of HI on day 1 on day 8 18 13 1.3 on day 15 6 4 20 1.56 on day 22 28 13 1.8 in day 43 24 11 5.9% of patients with the titer MN 80 on day 0 0 0 on day 9 9 1 on day 15 9 9 1 on day 22 12 9 1 on day 43 12 10 5 Geometric means of the title MN on day 1 10 10 10 on day 8 219 151 11 on day 15 1145 473 12 on day 22 375 324 12 on day 43 415 241 33 Relative ratio of MN on day 1 on day 8 22 15 1.1 on day 15 115 47 1.2 on day 22 37 32 1.2 on day 43 41 24 3.3 74

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SEQUENCE LISTING

&lt; 110 &gt; NOVARTIS AG

&lt; 120 &gt; VACCINATION WITH MULTIPLE CLADS OF H5 VIRUS FROM FLU A &lt; 130 &gt; P049768W0 &lt; 140 &gt; PCT / I82008 / &lt; 141 &gt; 2008-11-25 &lt; 150 &gt; US 61/004334 &lt; 151 &gt; 2007-11-26 &lt; 150 &gt; GB 0810305.3 &lt; 151 &gt; 2008-06-05 &lt; 160 &gt; 16 &lt; 170 &gt; SeqWin99, version 1.02

&lt; 210 &gt; 1 &lt; 211 &gt; 1707 &lt; 212 &gt; DNA &lt; 213 &gt; influenza A virus (A / Hong Kong / 213/03) &lt; 400 &gt; 1 atggagaaaa attggttacc actgttacac gatggagtga ccaatgtgtg ccagccaatg ttgagcagaa catgaagcct aggaatgtgg aataatacca gcagagcaga ctaaaccaga aggatggagt ggaaatttca atgaaaagtg ataaactcta tatgtgaaat agaagaagaa cagggaatgg gctgcagaca atcattgaca aggagaatag aatgctgaac gtcaagaacc aacggttgtt ggaacgtatg ggagtaaaat agttccctag tcgttacaat tagtgcttct atgcaaacaa atgcccaaga agcctctaat acgaattcat acctctgfcta taaaccattt cattaggggt tatggcttat accaagaaga ctaggctcta gattggtacc tcttctggac ttgctccaga aattggaata gtatgccatt caaacagatt aaaagagagg tagatggttg aagaatccac aaatgaacac agaatttaaa ttctggttct tttacgacaa tcgagttcta actacccgca tggagtcaat cactggcaat gcagaatttg ttttgcaata ctcgacagag catactggaa tttgagagat caatgtgccg cccaggggat tgagaaaatt gagctcagca caaaaagaac tcttttggta tcaaaaccca aaaaatagct aattttaaaa atatgcatac tggtaactgc ccacaatata agtccttgcg attatttgga gtatgggtac tcaaaaggca tcagtttgag caagaagatg catggaaaat ggtccgacta tcacaaatgt gtattcagaa aggaacttac catggtagct catttaa gtcagtcttg caggttgaca aagacacaca tgtagtgtag gaatggtctt ttcaacgact cagatcatcc tgtccatacc aatgcatacc accacctaca actagatcca ccgaatgatg ttgtggggga aaaactgtca aacaccaagt caccctctca actgggctca gctatagcag caccatagca atagatggag gccgttggaa gaagacggat gagagaactc cagcttaggg gataatgaat gaagcaagac caaatactgt ggtctatctt ttaaaagtga caataatgga acgggaagct ctggatggct acatagtgga atgaagaatt ccaaaaattc aaggaaagtc caacaataaa ttcaccatcc tttccgttgg aagtaaacgg caatcaactt agaaagggga gtcaaactcc ccatcgggga gaaatagccc gttttataga atgagcaggg tcaccaataa gggaatttaa tcctagatgt tagactttca ataatgcaaa gtatggaaag taaaaagaga caatttattc tatggatgtg tcagatttgc aaagaacgtt ctgcgatcta cctcggaaac gaaggccaat gaaacaccta ttggtccagt ctcctttttc gaggagctac taatgatgcg gacatcaaca gcaaaatgga cgagagcaat ctcageaatt aatgggggcg atgccccaaa tcaaagagag 9S9âggatgg gagtgggtac ggtcaactcg taacttagaa ctggacttat tgactcaaat ggagctgggt tgtaagaaac ggaaataagt tacagtggcg ctccaatggg 1 * i and 0 130 240 300 420 430 3BB S4D bQO BBO 720 760 340 * 100 ** bO 1020 1030 1140 1200 121.0 1320 1330 1440 1500 lSbO l b2Q lb30 1707 &lt; 210 &gt; 2

&lt; 211 &gt; 17.07 &lt; 212 &gt; DNA &lt; 213 &gt; influenza virus A (A / Indonesia / 5/05) &lt; 400 &gt; 2 atggagaaaa attggttacc actgttacac tagtgcttct atgcaaacaa atgcccaaga tcttgcaata ttcaacagag catactggaa gtcagtcttg caggttgaca aagacacaca ttaaaagtga caatcatgga acgggaagct tcagatttgc aaagaacgtt ctgcgatcta bQ 120 130 86 gatggagtga agcctetaat ttfcaagagat tgtagtgtag ctggatggct cctegggaac 210 ccaatgtgtg acgaattcat caatgtaccg gaatggtctt acatagtgga 300 gaaggccaat

ccaaccaatg acctctgtta cccagggagt ttcaacgact atgaagaact gaaacaccta 3BQ ttgagcagaa taaaccattt tgagaaaatt caaatcatcc ccaaaagttc ttggtccgat 4 20 catgaagcct catcaggagt gagctcagca tgtccatacc tgggaagtcc ctcctttttt 430 agaaatgtgg tatggcttat caaaaagaac agtacatacc caacaataaa 540 gaaaagctac

aatatacca accaagaaga tcttttggta ctgtggggaa ttcaccatcc taatgatgcg bOQ

gcagagcaga caaggctata tcaaaaccca accacctata tttccattgg gacatcaaca bbQ ctaaaccaga gattggtacc aaaaatagct actagatcca aagtaaacgg gcaaagtgga 720 aggatggagt tcttctggac aattttaaaa cctaatgatg caatcaactt cgagagtaat 730 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcagcaatt 340

atgaaaagtg aattggaata tggtaactgc aacaccaagt gtcaaactcc aatgggggcg 'JQQ

ataaactcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa 'JbO tatgtgaaat caaacagatt agtccttgca acagggctca gaaatagccc tcaaagagag 1020 agcagaagaa aaaagagagg actatttgga gctatagcag gttttataga gggaggatgg 1020

cagggaatgg tagatggttg gtatgggtac caccatagca atgagcaggg gagtgggtac UfO gctgcagaca aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactca 1200

atcattgaca aaatgaacac tcagtttgag gccgttggaa gggaatttaa taacttagaa 12bO aggagaatag agaatttaaa caagaagatg gaagacgggt ttctagatgt ctggacttat 1320 aatgccgaac ttctggttct catggaaaat gagagaactc tagactttca tgactcaaat 1330 gttaagaacc tctacgacaa ggtccgacta cagcttaggg ataatgcaaa ggagctgggt 1440 aacggttgtt tcgagttcta tcacaaatgt gataatgaat gtatggaaag tataagaaac 1500 ggaacgtaca actatccgca gtattcagaa gaagcaagat taaaaagaga 15b0 ggaaataagt

ggggtaaaat tggaatcaat aggaacttac caaatactgt caatttattc aacagtggcg lb2Q

agttccctag cactggcaat catgatggct ggtctatctt tatggatgtg ctccaatgga IbfiO tcgttacaat gcagaatttg catttaa 1707 &lt; 210 &gt; 3 &lt; 211 &gt; 1654

&lt; 212 &gt; DNA &lt; 213 &gt; influenza A virus (A / Chicken / Hong Kong / SF219 / 01) &lt; 400 &gt; 3

agtcttgtta aaagtgatca gatttgcatt ggttaceatg caaacaactc gacagagcag bQ gttgacacaa taatggaaaa gaacgttact gfctacacatg cccaagacat attggaaaag 120

acacacaatg ggaagctctg cgatctagat ggagtgaagc ctctaatttt gagagattgt lâO agtgtagctg gatggctcct cggaaaccca atgtgtgacg aattcatcaa tgtgccggaa 240 tggtcttaca tagtggagaa ggceagtcca gccaatgacc tctgttaccc aggggatttc 300

aacgactatg aagaactgaa acacctattg agcagaataa accattttga gaaaattcag 3bD

atcatcccca aaagttcttg gtccaatcat gaagcctcat caggggtgag ctcagcatgt M2D ccataccttg ggaagtcctc ctttttcaga aatgtggtat ggçttatcaa aaagaacagt 430 acatacccaa caataaagag gagctataat aataccaacc aagaagatct tttggtactg 540

tgggggattc accatcctaa tgatgcggca gagcagacaa agctctatca aaacccaacc bOO

acctatattt ccgttggaae atcaacacta aaccagagat tggtaccaaa aatagctact BBO agatccaaag taaacgggca aagtggaaga atggagttct tctggacaat tttaaagccg 720 aatgatgcta tcaatttcga gagtaatgga aatttcattg ctccagaata tgcâtacaaa 730 attgtcaaga aaggggactc atcaattatg aaaagtgaafc tggaatatgg taaefcgeaac 340 accaagtggc aaactccaat aaetctagta tgccattcca caacatacac gggggcgata * I00

cctctcacca tcggggaatg ccccaaatat gtgaaatcaa acagattagt ccttgcgact 4BQ ggactcagaa atacccctca aagagagaga agaagaaaaa agagaggact atttggagct 1020 atagcaggtt ttatagaggg aggatggcag ggaatggtag atggttggta tgggtaccac 1030 agcaggggag tggatacgct gcagacaaag catagcaatg aatccactca aaaggcaata 1140 gatggagtta ccaataaggt caactcgatc attgacaaaa tgaacactca 1200 gtttgaggcc

gttggaaggg aatttaataa cfctagaaagg agaatagaaa atttaaacaa gaagatggaa 12bQ gacggattcc tagatgtctg gecttataat gctgaacttc tggttctcat ggaaaatgag 1320 agaactctag actttcacga ctcaaatgtc aagaaccttt acgaeaaggt ccgaetacag 1330 cttagggata atgcaaagga.gctgggtaae ggctgtttcg agttctatca caaatgtgat 1440 aatgaatgta tggaaagtgt aaaaaacgga acgtatgact acecgeagta 1500 ttcagaagaa

gcaagactaa acagsgagga aataagtgga gtaaaattgg aatcaatggg aacttaccaa 15bQ

atactgtcaa tttattcaac agtggcgagt tccctageac tggcaatcat ggtagctggt lb2Q ctatctttat ggatgtgcte caatggatcg ttac lb54 87

&lt; 210 &gt; 4 <211> 1668 <212> DNA &lt; 213 &gt; influenza virus A (A / chicken / Guiyang / 441/2006) &lt; 400 &gt; 4 atggagaaaa tagtgcttct tcttgcaata attggttacc atgcaaacaa ctcgacagtg actgtaacac atgcccaaga catactggaa gatggagtga agcctctaat tttaagagat ccaatgtgtg acgaattcat caatgtgccc ccagccaatg acctctgtta cccaggggat ttgagcagaa taaaccattt tgagaaaatt catgaagcct cactaggggt gagctcagca agaaatgtgg tatggcttat caaaaagaac aataatacca accaagaaga tcttttagta gcagagcaga taaagcttta tcaaaaccca ctaaaccaga ggttggtacc aacaatagct aggatggagt tcttctggac aattttaaag ggaaatttca ttgctccaga atatgcatac atgaaaagtg aattggaata tggtaactgc ataaactcta gtatgccatt ccacaacata tatgtgaaat caaacagatt agtccttgca agaaggagaa aaaagagagg acfcatttgga cagggaatgg tagacggttg gtatgggtac gctgcagaca aagaatccac tcaaaaggca atcattaaca aaatgaacac tcagtttgag aggagaatag aaaatttaaa caagaagatg aatgctgaac ttctggttct catggaaaat gtcaagaacc tttacgacaa agtccgacta aacggttgtt tcgagttcta tcacaaatgt ggaaegtatg actacccgca gtatteagaa ggagtaaaet tggaatcaat gggaacctac agttccctag cactggeaat catggtagct atcagtcttg ttaaaagtga tcagatttgc caggttgaca cgataatgga aaagaatgtt aagaeac ACA atgggaagct ctgcagtcta tgtagtgtag ctggatggct cctcggaaac gaatggtctt acatagtgga gaaggccagt ttcaacgact acgaagaact gaaacaccta cagatcatcc ccaaaagttc ttggcccaat tgtccatacc tgggggagtc ctcctttttc agttcatacc caacaataaa gaggagctac ttgtggggga tccatcaccc taatgatgcg aacacctata tttccgttgg aacatcaaca actagatcca aagtaaacgg gcaaagtgga ccgaatgata ctatcaattt cgagagtaat aaaattgtca agaaagggga ctcagcaatt aacaccaagt gtcaaactcc aatgggggcg caccctctca ccatcgggga atgccccaaa actggactca gaaataccct tcaaagagag gccatagcag gttttattga gggaggatgg caccatagca atgagcaggg gagtggatac atagatggaa tcaccaataa ggtcaactcg gccgttggac gggaatttag taacttagaa gaagacggat tcctagatgt ctggacttat gaaagaactc tagactttca tgactcaaat cagcttaggg ataatgcaaa agagctgggt gatgatgaat gtatggaaag tgtaaaaaac gaagctagac taaacagaga ggagataaat caaatactgt caatttactc aacagtggcg ggtctatctt tatggatg bO ISO 180 240 300 31 * 0 430 430 S40 (&gt; 00 (&gt; {&gt; 0? 30? 840 <100 1030 1080 1140 1300 131.0 1330 1380 1440 1500 151.0 11.30 1U.8

<210> 5 <211> 1698 <212> DNA &lt; 213 &gt; influenza virus A (A / duck / Guangxi / 1681/2004) &lt; 400 &gt; 5 88 atggagaaaa attggttacc actgttacac gatggagtga ccaatgtgtg ccagccaatg ttgagcagaa catgaagect aggaatgtgg sataatacca gcagagcaga ctaaaccaga agaatggagt ggaaatttca atgaaaagtg ataaactcta tatgtgaaat ataagaagaa cagggaatgg gctgcagaca atcattgaca aggagaatag aatgctgaac gtcaagaaec aacggttgtt ggaacgtatg ggagtaaaat tagtgcttct atgcaaacaa atgcccaaga agcctctaat atgaattcat acctctgtta taaatcattt catcaggggt tatggcttat gtcaagaaga caaagctcta gatfcggtacc tcttctggac ttgctccaga aattggaata gtatgccafct caagcagatt aaaagagagg tagatggttg aagaatccac aaatgaacac agaatttaaa ttctggttct tttacgacag tcgagtteta actacccgea tggaatcgat tcttgcaate ctcgacagag catactggaa tttgagagat caatgtgccg cccaggagat tgagaaaafct gagctcagca caaaaagaac tcttttggta tcaaaaccca aaaaatagct aattttaaag atafcgcctac tggtaactgc ccacaacata agtccttgcg actatttgga gtatgggtac tcaaaaggca tcaatttgag caaaaagatg catggaaaat ggtccgacta tcacaagtgt gtattcagaa aggaacttac gtcagtcttg caggttgata aagacacaca tgtagtgttg gaatggtctt ttcaacgact cagatcatcc tgtccatacc c agtgcatacc tgtggggga actacctata actagatcca ccgaatgatg aaaattgfcca aacaccaaat caccctctca acagggctca gctatagcag caccatagca atagatggag gccgttggaa gaggacggat gagagaactc cagcttaggg gacaatgaat gaagcaagac caaatattgt ttaaaagtga caataatgga acgggaaact cgggatggct acatagtgga atgaagaact ccaaaagttc aggggaggcc ctacaataaa ttcaccatcc tttcegttgg aagtaaacgg ctatcaactt agaaagggga gtcaaactcc ccatcgggga ggaatagccc gctttataga acgagcaggg tcaccaataa gggaatttaa tcctagatgt tagactttca ataatgcaaa gcatggaaag taaagagaga caatttattc tcagatttgc aaagaatgtt ctgcgatcta cctcggaaac gaaggccagt aaaacaccta ttggtccaat ctcctttttc gaggagctac taatgatgcg aacatcaaca gcagagtgga cgagagtaat ctcagcaatt aatgggggcg atgccccaag tcaaagagag gggaggatgg gagtggatac agtcaattcg taatttagaa ctggacttat tgactcaaat ggagctgggg tgtaagaaac ggaaataagt aacagtggcg

to ISO IflQ 5MQ 300 3tO 420 4OQ 540 too% A0 S40 400% Q 1020 1000 114G ie this 1330 13fiQ 1440 1500 15t0 lb2D agttccctag cactggcaat catggtagct tcgttaeaat gcagaatt ggtctatctt tatggatgtg ctccaatgga

IbôQ lb4â

&lt; 210 &gt; 6 &lt; 211 &gt; 1707 &lt; 212 &gt; DNA &lt; 213 &gt; influenza A virus (A / tree sparrow / Henan / 4/2004) &lt; 40 0 &gt; 6 89

atggagaaaa tagtgcttet tcttgcaata gtcagtcttg ttaaaagtga tcagatttgc 1Q

attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgca aaagaacgtt ISO actgttacac atgcccaaga catactggaa aagacacaca acgggaagct ctgcgatcta 160 gatggagtga aacctctaat tttaagagat tgtagtgtag ctggatggct cctcggaaac 2 * 10 ccaatgtgtg aegaattcat caatgtgccg gaatggtctt acatagtgga gaaggccagt 300 ccagccaatg acctctgtta cccaggggat ttcaacgact atgaagaact gaaacaeeta 310 ttgagcagaa taaaccattt tgagaaaatt cagatcatcc ccaaaagttc ttggtccgat ** 20 catgaagcct eatcaggggt gagctcagca tgtccatacc aggggagtcc ctcctttttc 460 agaaatgtgg tatggcttat caaaaagaac agtgcatacc caacaataaa gaggagctac 540

aataataeca accaagaaga tcttttggta ctgtggggga ttcaccatcc taatgatgcg fcOQ

gcagagcaga tcaaaaccca aecaeetata tttccgttgg caaagctcta aacatcaaca TBO ctaaaccaga gattggtacc aaaaatagct actagatcca aagtaaatgg gcaaagtgga 720 agaatggagt tcttctggac aattttaaag ccgaatgacg ctatcaactt cgagagtaat 760 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcagcaatt 640 atgaaaagtg aattggaata tggtaactgc aacaccaagt gtcaaactcc 100 aatgggggcg

ataaactcta gtatgccatt ccacaacata caecctetca ccategggga atgccccaaa ITO tatgtgaaat caaacagatt agtecttgcg acagggctca gaaatagccc tcaaagagag 1020 agaagaagaa aaaagagagg actatttgga gctatagcag gttttataga gggaggatgg 1030 cagggaatgg tagatggttg gtatgggtac caccatagca atgagcaggg gagtggatac 1140 gctgcagaca aagaatccac tcaaaaggca atagatggag teaeeaataa ggtcaactcg 1200 atcattgaca aaatgaacac tcagtttgag gccgttggaa gggaatttaa taacttagaa 12t0 aggagaatag aaaatttaaa caagaagatg gaggacggat tcctagatgt ctggacttat 1320 aatgctgaac ttctggttct catggaaaat gagagaacte tagactttca tgactcaaat 1380 gtcaagaacc tttacgaaaa ggtccgacta caacttaggg ataatgcaaa ggagctgggt 1440 aacggttgtt tcgagttcta tcacaaatgt gataatgaat gtatggaaag tgtaaaaaac 1500 ggaacgtatg actacccgca gtattcagaa gaagcaagac taaacagaga ggaaataagt 1510 ggagtaaaat tggaatcaat aggaacttac caaatactgt caatttattc 1120 aacagtggtg

agttccctag eactggcaat catggtagcfc ggtctatctt tatggatgtg ctccaatgga IbfiO tcgttacaat gcagaatttg catttaa 1707

&lt; 210 &gt; 7 &lt; 211 &gt; 1707 &lt; 212 &gt; DNA &lt; 213 &gt; influenza virus A (A / chicken / Shanxi / 2/2006) &lt; 400 &gt; 7

atggagaaaa tagtgcttet tcttgcaata atcggtcttg ttaaaagtga tcagatttgc bO gttggttacc atgcaaacaa ctcaacagag caggttgaca caataatgga aaagaacgtt 120 actgttacac atgctcaaga catactggag aagacacaca acgggaagct ctgcgaccta 180 gatggagtga agcctctcat tttgaaagat tgtagtgtag ctggatggct cctcggaaac 240 cctatgtgtg acgaatttat caatgtgtcg gaatggtctt acatagtgga 300 gaaggccagt

ccagccaatg gcctctgtta cccaggggat ttcaatgact atgaagaact gaaacaccta 3BD ttgagcagaa taaaccattt tgagaaaatt aagatcatcc ccaaaagttc ttggtccaat 420 catgaagcct eatcaggggt gagctcagca tgttcctatc tggggaagcc ctcctttttc 450 agaaatgtgg tatggcttat caaaaagaat aatacatacc caccaataaa 540 ggtgaactac

accaatacca accaagaaga tcttttggta ctgtggggga ttcaccatcc caacgatgag bOO

acagagcaga taaagatcta tcaaaaccca accacctata tttccgttgg aacatcaaca bbO ctaaaccaga gattggtacc aaaaatagct actaaatccc aagtgaacgg gcaaagtgga 720 agaatggagt tcttctggac aattttaaag ccgaatgatg ctatcaattt cgatagtaat 780 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga eteagegatt 640

atgaaaagtg aattggaata tggtaactgc aacaccaagt gtcaaactcc aatgggggcg *) 0D

ataaattcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa SBQ tatgtgaaat caaacagatt agtcctcgcg actggactca gaaatgcccc tcaaagagag 1020 ggaagaagaa aaaagagagg actatttgga gccatagcag ggtttataga gggaggatgg 1060 cagggaatgg tagatggttg gtatgggtac caccatagca atgagcaggg gagtggatac 1140 tctgcagaca aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg 1200 90 atcattgaca aaatgaacac tcagtttgag aggagaatag agaatttaaa caaaaagatg aacgctgaac ttctggttct catggaaaat gtcaagaacc tttacgacaa ggtccgactg aacggttgtt tcgaattcta tcacaaatgt ggaacgtatg actacccgca gtattcagaa ggagtaaaat tggaatcaat ggtaacttac agttccctag cattggcaat catggtggct tcgttacaat gcagaatttg catttga ggegttgtfca gggaaattaa taacttagaa gaagacggat tcctagatgt ctggacttaC gagagaactc tagactttca tgactcaaat cagcttaggg ataatgcaaa ggagctgggt gataatgaat gtatggaaag tgtaaaaaac gaagcaagac taaacagaga ggaaataagt caaatactgt caatttattc aacagtggcg ggtctatctt tatggatgtg ctccaatgga 12bQ 1320 1380 1440 1500 15b0 lb20 lbSD l Q7 &lt; 210 &gt; 8

&lt; 211 &gt; 1707 &lt; 212 &gt; DNA &lt; 213 &gt; influenza virus A (A / Chicken / Henan / 12/2004) &lt; 400 &gt; 8 atggagaaaa attggttacc actgttacac gatggagtga ccaatgtgtg ccagccaatg ttgagcagaa catgaagcct agaaatgtgg aataatacca gcagagcaga ctaaaccaga aggatggagt ggaaatttca atgaaaagtg ataaactcta tatgtgaaat agaagaagaa cagggaatgg gctgcagaca atcattgaca aggagaatag aatgctgaac gtcaagaacc aacggttgtt ggaacgtatg ggagtaaaat agttccctag tcgttacaat tagtgcttct atgcaaacaa atgcccaaga agcctctaat acgaattcat gcctctgtta taaaccattt catcaggggt tatggcttat accaagaaga caaggctcta gattggtacc tcttctggac ttgctccaga aattggaata gtatgccatt caaacagatt aaaagagagg tagatggttg aagaatccác aaatgaacac agaatttaaa ttctggttct tttacgacaa tcgagttcta actacccgca tggaatcaat cactggcaat gcagaatttg tcttgcaata ctcgacagag catactggaa tttgagagat caatgtgccg cccaggggat tgagaaaatt gagctcagca caaaaagaac tcttttggta tcaaaaccca aaaaatagct aattttaaaa atatgcatac tggtaactgc ccacaacata agtccttgcg actatttgga gtatgggtac tcaaaaggca tcagtttgag caagaagatg catggaaaat ggtccgacta tcacaaatgt gtattcagaa aggaacttac catggtagct catttaa gtcagtcttg caggttgaca aagacacaca tgtagtgtag gaatggtctt ttcaacgact cagatcatcc tgcceatacc agtacatacc ctgtggggga accaectata actagatcca ecgaatgatg aaaattgtca aacaccaagt caccctctca actgggctca gctatagcag caccatagca atagatggag gccgttggaa gagagaactc cagcttaggg gataatgaat gaagacggat gaagcaagac caaatactgt ggtctatctt ttaaaagtga teagatttgc caataatgga aaagaacgtt acgggaagct ctgcgatcta ctggatggct cctcggaaac acatagtgga gaaggccagt atgaagaact gaaacsccta ccaaaagttc ttggtccaat agggaaagtc ctcctttttc caacaataaa gaggagctac ttcaccatcc taatgatgeg tttccgttgg aacatcaaca aagtaaacgg gcaaagtgga caatcaactt cgagagtaat agaaagggga ctcagcaatt gtcaaactcc aatgggggca ccatcgggga atgccccaaa gaaatagccc tcaaagagag gttttataga gggaggatgg atgagcaggg gagtggatac tcaccaataa ggtcaaetcg gggaatttaa taacttagaa tcctagatgt ctggacttat tagactttca tgactcaaat ataatgcaaa ggagctgggt gtatggaaag tgtaagaaac taaaaagaga ggaaataagt caatttattc aacagtggcg tatggatgtg ctccaatgga LD 120 180 240 300 3B0 420 480 540 BBQ BBO 720 780 840 100 *? b0 1020 1080 1140 1200 IHbO 1320 1380 1440 1500 ISbO lb2Q 210> 9 <211> 1695 <212> DNA &lt; 213 &gt; influenza A virus (A / duck / Guangxi / 2775/2005) &lt; 400 &gt; 9 91 atggagaaaa tagtgcttct attggttacc atgcaaacaa actgttacac atgcccaaga gatggggtga agcctctaat ccaatgtgtg acgaattcat ccagceaatg acctctgtta ttgagcagaa taaaccattt catgaagcct catcaggggt agaaatgtgg Catggcttat aataatacca accaagaaga acagagcaga caaagctcta ctaaaccaga gattggtacc aggatggagt tcttctggac tcttgcaata gtcagtcttg ctcgacagag caggttgaca catactggaa aagacacaca tttgagagat tgtagtgtag caatgtaçcg gaatggtctt cccaggcgat ttcaacgatt tgagaaaatt cagatcatcc gagctcagca tgtccatacc caaaaagaac agtgcatacc tcttttggta ctgtggggga tcaaaaccca accacttata aaaaatagct accagatcca aattttaaaa ccgaatgatg ttaaaagtga tcagatttgc caataatgga aaagaacgtt atgggaagct ctgcgaecta ctggatggct cctcgggaac acatagtgga gaaggccagt atgaagaact gaaacaccta ccaaaagttc ttggcccaac tgggaaagcc ctcctttttc caaeaataaa gaggagctac ttcaccatcc taatgatgag tttccgttgg aacatcaaca aagtaaacgg gcaaagtgga caatcaactt cgagagtaat t '0120 ISO 240 300 3BQ 420 460 S4Q bOO BBO 720 7S0 ggaaatttca ttgctccaga atatgcctac aaaattgtca agaaagggga ctcagcaatt atgaaaagtg aattgg AATA tggtaactgc aacaccaaat gtcaaactcc aatgggggcg ataaactcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa tatgtgaaat caaacagatt agtccttgcg actgggctca gaaatagecc tcaaagggag agaagaagaa aaaagagagg actatttgga gctatagcag gttttataga gggaggatgg cagggaatgg tagatggttg gtacggatac caccatagca atgagcaggg gagtggatac gctgcagaca aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg atcattgaca aaatgaacac tcagtttgag gccgttggaa gggaatttaa taacttagaa aggagaatag agaatttaaa caagaagatg gaagacgggt tectagatgt ctggacttat aatgctgaac ttctggttct catggaaaat gagcgaactc tagactttca tgactcaaat gtcaagaaec tttacgacaa ggtccgacta cagctfcaggg ataatgcaaa agagctgggt aacggttgtt tcgagttcta tcacaaatgt gataatgaat gcafcggaaag tgtaagaaac ggaacgtatg actacccgca ttattcagaa gaagcaaggc taaaaagaga ggaaataagt ggagtaaaat tggaatcaat aggaacttac caaatactgt caatttattc aacagtggca agttccctag cgctggcaat catggtagct ggtctatctt tatggatgtg ctccaatggt tcgttacaat gcaga

&lt; 210 &gt; 10 &lt; 211 &gt; 1707 &lt; 212 &gt; DNA &lt; 213 &gt; influenza virus A (A / Hong Kong / 156/97) &lt; 4 0 0 &gt; 10640400 UQ 1020 1060 1140 1200 121.0 1320 1360 1440 1500 ISfeO 1É.20 IbãO lt.45 92 atggaaagaa attggttacc actgttacac aatggagtga cctatgtgtg ccagccaatg ttgagcagaa catgatgcct agaaatgtgg aataatacca gcagagcaga ctgaaccaga agaatggagt ggaaatttca atgaaaagtg ataaactcta tatgtgaaat agaagaagaa cagggaatgg tctgcagaca atcattaaca aggaggatag aatgctgaac gtcaagaacc aatggttgtt ggaacgtatg ggagtaaaat agttccctag tcgttacaat cagtgcttct atgcaaacaa atgcccaaga agcctctcat acgaattcat acctctgtta taaaccattt catcaggggt tatggcttat accaagaaga caaagctcta gattggttcc tcttctggac ttgctccaga aattggaata gtatgccatt caaacagatt tagatggttg aagaatccac aaatgaacac agaatttaaa ttctggttct tttacgacaa ccgaattcta actacccgca tggaatcaat cactggcaat gcagaatttg tcttgcaaca ctcgacagag catactggaa tttgagggat caatgtgccg tccagggaat tgagaeaatt gagctcagca caaaaagaac tcttttggta tcaaaatcca agaaatagct aattttaaag atatgcatac tggtaactgc ccacaacata agtccttgcg actatttgga gtatgggtac tcaaaaggca tcagtttgag caagaagatg catggaaaat ggtccgacta tcacaaatgt gtattcagaa gggaacttac catggtagct catttaa gtcagtcttg caggttgaca aggacacaca tgtagtgtag gaatggtctt ttcaacgact cagatcatce tgtccatacc agtgcatacc ctgtgggggg accacctaca actagaccca ccgaatgatg aaaattgtca aacaccaagt caccccctca actggactca gctatagcag caccatagca atagatggag gccgttggaa gaagacggat gagagaactc cagcttaggg gátaatgaat gaagcaagac caaatactgt ggtctatctt ttaeaagtga caataatgga acgggaagct ctggatggct acatagtgga atgaagaact ccaaaagttc ttgggaggtc caacaataaa ttcaccatcc tttccgttgg aagtaaacgg ccatcaattt agaaagggga gtcaaactcc ccatcgggga gaaatacccc gttttataga atgagcaggg tcaccaataa gggaatttaa tcctagatgt tcgactttca ataatgcaaa gtatggaaag taaacagaga caatttattc tatggatgtg tcagatttgc aaagaatgtt ctgcgatcta cctcggaaac gaaggccagt gaaacaccta ttggtccaat ctcctttttc gaggagctac taatgatgcg aacatcaaca gcaaagtgga cgagagtaat ctcaacaatt aatgggggcg atgccccaaa tcaaagagag gggaggatgg gagttgctac ggtcaactcg taacttggaa ctggacttac tgactcaaat ggagctgggt tgtaaaaaac ggaaataagt aacagtggeg ctccaatgga bQ 120 ion 2 * 40 300 3B0 &gt; 420 * 4d SHO b OO bbO 720 760 Ego ISO 1010 a 11 11 40 40 12 12 13 0 13 SQ 1 * 4 * 40 1SQ0 15bQ lb 20 Ib 10 1707

&lt; 210 &gt; 11 &lt; 211 &gt; 1704 &lt; 212 &gt; DNA &lt; 213 &gt; influenza virus A (A / Anhui / 1/2005) &lt; 400 &gt; 11 atggagaaaa tagtgcttct attggttacc atgcaaacaa actgttacac atgcccaaga gatggagtga agcctctgat ccaatgtgtg acgaattcat ccagccaatg acctctgtta tcttgcaata gtcagccttg ctcgacagag caggttgaca catactggaa aagacacaca tttaagagat tgtagtgtag caatgtgccg gaatggtctt cccagggaat ttcaacgact ttaaaagtga tcagatttgc caataatgga aaagaacgtt acgggaagct ctgcgatcta ctggatggct cctcggaaac acatagtgga gaaggccaae atgaagaact gaaacaccta bO 120 160 2M0 3DQ 3B0 93 ttgagcagaa taaaccattt tgagaaaafct cagatcatcc ccaaaagttc ttggtccgat 430 catgaagcct catcaggggt gagctcagca tgtccatacc agggaacgcc ctcctttttc 4S0 agaaatgtgg tatggcttat caaaaagaac aatacatacc caacaataaa gagaagctac 540 aataatacca accaggaaga tcttttgata ctgtggggga ttcatcattc taatgatgcg 500 gcagagcaga caaagctcta tcaaaaccca accacctata tttccgttgg gacatcaaca 550 ctaaaccaga gattggtacc actagatcca aagtaaacgg gcaaagtgga 720 aggatggatt aaaaatagct tcttctggac aattttaaaa ccgastgatg caatcaactt cgagagtaat 7S0 ggaaatttca ttgctccaga atatgcatae aaaattgtca agaaagggga ctcagcaatt O40 gttaaaagtg aa gtggaata tggtaactgc 400 aacacaaagt gtcaaactcc aataggggcg ataaactcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa 450 tatgtgaaat caaacaaatt agtccttgcg actgggctca gaaatagtcc tctaagagaa 1020 agaagaagaa aaagaggact atfctggagct atagcagggt ttatagaggg aggatggcag 1QS0 ggaatggtag atggttggta tgggtaccac catagcaatg agcaggggag 1140 tgggtacgct

gcagacaaag aatccactca aaaggcaata gatggagtca ccaataaggt caactcgatc 12DO attgacaaaa tgaacactca gtttgaggcc gttggaaggg aatttaataa cttagaaagg 1250 agaatagaga atttaaaeaa gaaaatggaa gacggattcc tagatgtctg gaettataat 1220

gctgaacttc tggttctcat ggaaaatgag agaactctag acttccatga ttcaaatgtc 13SD ggttgtttcg agttctatca caaatgtgat aatgaatgta tggaaagtgt aagaaacgga 1500 acgtatgact acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1550 gtaaaattgg aatcaatagg aacttaccaa atactgtcaa tttattcaac agttgcgagt 1520 tctctagcac tggcaatcat ggtggctggt ctatctttgt ggatgtgctc caatgggtcg 1580 ttacaatgca gaatttgcat ttaa 1704 aagaaccttt acgacaaggt ccgactacag ctfcagggata atgcaaagga 1440 gcCgggtaac

&lt; 210 &gt; 12 &lt; 211 &gt; 1707 &lt; 212 &gt; DNA &lt; 213 &gt; influenza A virus (A / turkey / Turkey / 1/05) &lt; 4 0 0 &gt; 12 atggagaaaa tagtgcttct tcttgcaata gtcagccttg ttaaaagtga tcagatttgc 50 attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtc 120

actgttacac acgcccaaga catactggaa aagacacaca acgggaaact ctgcgatcta ISO gatggagtga agcctctaat tttaagagat tgtagtgtag ctggatggct cctcgggaac 240 ccaatgtgtg acgaattcct caatgtgccg gaatggtctt acatagtgga gaagatcaat 300 ccagccaatg acctctgtta cccagggaat ttcaacgact atgaagaact gaaacaccta 350 ttgagcagaa taaaccattt tgagaaaatt cagatcatcc ccaaaagttc ttggtcagat 420 catgaagcct cagcaggggt gagctcagca tgtccatacc agggaaggtc ctcctttttt 4S0 agaaatgtgg tatggcttat caaaaaggac aatgcatacc caacaataaa gagaagttac 540 aataatacca accaagaaga tcttttggta ttgtggggga ttcaccatcc aaatgatgcg 500 gcagagcaga caaggctcta tcaaaaccca actacctata tttccgttgg gacatcaaca 550 ctaaaccaga gattggtacc aaaaatagcc actagatcta aggtaaacgg gcaaagtgga 720 aggatggagt tcttttggac aattttaaaa ccgaatgatg caataaactt tgagagtaat 7S0 ggaaatttca ttgctccaga aaatgcatac aaaattgtca agaaagggga ctcaacaatt S40 atgaaaagtg agttggaata tggtaactgc aacaccaagt gtcaaactcc aataggggcg 400 ataaactcta gtatgccatt ccacaacatc caccctctca ccatcgggga atgccccaaa * 150 tatgtgaaat caagcaga tt agtccttgct actgggctca gaaatagccc tcaaggagag 1020 agaagaagaa aaaagagagg actatttgga gctatagcag gttttataga gggaggatgg 10S0 cagggaatgg tagatggttg gtatgggtac caccatagca acgagcaggg gagtgggtac 1140 gctgcagaca aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg 1200 atcattgaca aaatgaacac tcagtttgag gctgttggaa gggaatttaa taacttagaa 1250 aggagaatag aaaatttaaa caagaagatg gaagacggat tcctagatgt ctggacttat 1320 aatgctgaac ttctggttct catggaaaat gagagaactc tagactttca tgactcaaat 13S0 gtcaagaacc tttacgacaa ggtccgacta cagcttaggg ataatgcaaa ggagcttggt 1440 ggaacgtatg actacccgca gtattcagaa gaagcaagat taaaaagaga ggaaataagt 1550 ggagtaaaat tggaatcaat aggaacttac caaatactgt caatttattc aacagtggcg 1520 agctccctag cactggcaat catggtggct ggtctatctt tatggatgtg ctccaatgga 15S0 tcgttacaat gcagaatttg catttaa 170? aacggttgtt tcgagttcta tcacagatgt gataatgaat gtatggaaag tgtaagaaac 1500 94

<210> 13 <211> 8 <212> PRT <213> influenza virus A &lt; 4 0 0 &gt; 13

Arg filu Gly Arg Arg Arg Lys Arg 1 S

<210> 14 <211> 2 <212> DNA <213> Artificial Sequence &lt; 22 0 &gt; &lt; 223 &gt; oligonucleotide Adjuvant &lt; 22 &gt;

&lt; 221 &gt; N &lt; 222 &gt; 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 &lt; 223 &gt; N is inosine &lt; 400 &gt; 14 ncncncncncncncncncncncncnc 26

<210> 21 <211> 11 <212> PRT <213> Artificial Sequence &lt; 22 0 &gt; &lt; 223 &gt; polycationic peptide adjuvant &lt; 4 0 0 &gt; 15 95

Lys Leu Lys Leu Leu Leu Leu Leu Lys Leu Lys 1 S 10

<210> <211> <213> <213> influenza virus A &lt; 4 0 0 &gt; 16

Gly Glu Arg Arg Arg Arg Lys Arg 1,596

Claims (18)

An immunogenic composition comprising the hemagglutinin antigen of a first influenza A H5 virus clade for use in a method for immunizing a subject against influenza virus comprising the steps of (i) administering to the subject the immunogenic composition comprising the hemagglutinin antigen from the first clade of influenza A H5 virus, and then (ii) administering to a subject an immunogenic composition comprising the hemagglutinin antigen of a second clade of influenza A H5 virus, wherein the first and second clades are different from each other, and wherein: (a) the composition comprising the first clade antigen is adjuvanted and / or the composition comprising the second clade antigen is adjuvanted; (b) the antigen from the first clade and / or from the second clade comprises neuraminidase from the H5 virus, (c) the first clade is not clade 0; or (d) the composition comprising the first clade antigen contains 0.1-15æg haemagglutinin of the first H5 clade of influenza A virus and / or the composition comprising the second clade antigen contains 0.1-15pg haemagglutinin of the second clade of influenza A virus. An immunogenic composition comprising the hemagglutinin antigen of a second influenza A H5 virus clade for use in a method for immunizing a subject against influenza virus comprising the steps of (i) administering to a subject an immunogenic composition comprising the hemagglutinin antigen of a first clade of influenza A H5 virus, and then (ii) administering to the subject an immunogenic composition comprising the hemagglutinin antigen of the second clade of influenza A H5 virus, wherein the first and second clades are different, and wherein: (a) the composition comprising the first clade antigen is adjuvanted and / or the composition comprising the second clade antigen is adjuvanted; (b) the antigen from the first clade and / or from the second clade comprises neuraminidase of the H5 virus, (c) the first clade is not clade 0; or (d) the composition comprising the first clade antigen contains 0.1-15æg haemagglutinin of the first clade of influenza A H5 virus and / or the composition comprising the second clade antigen contains 0.1-15pg haemagglutinin second clade of influenza A H5A virus. The immunogenic composition of claim 1 or 2, wherein: • the first clade is clade 1 and the second clade is clade 2. • the first clade is clade 2 and the second clade is clade 1. • the first clade is the clade 1 and the second clade is not the clade 2. • the first clade is the clade 2 and the second clade is not the clade 1. The immunogenic composition of any preceding claim, wherein at least one of the immunogenic compositions is a fragmented virion virus. The immunogenic composition of any preceding claim, wherein at least one of the immunogenic compositions includes influenza A virus neuraminidase. The immunogenic composition of any preceding claim, wherein the composition comprising the first antigen of the clade is adjuvanted. The immunogenic composition of any preceding claim, wherein the composition comprising the second clade antigen is adjuvanted for example wherein the composition comprising the first clade antigen is adjuvanted and the composition comprising the second clade antigen is adjuvanted. An immunogenic composition comprising hemagglutinin antigens of more than 5 influenza A virus clade. The composition of claim 8, comprising hemagglutinin of influenza A H5 virus clades 1 and 2. The composition of claim 8 or claim 9, wherein the composition is as adjuvanted (for example, with a submicron oil-in-water emulsion). The composition of any one of claims 8 to 10, wherein the composition contains between 0.1 and 50æg of haemagglutinin per H5 clade. The composition of claim 11, wherein the composition contains 0.1 - 15pg of haemagglutinin per H5 clade, for example 7.5pg per clade or 3.75pg per clade. The composition of any one of claims 10 to 12, which contains the whole virion antigens and an aluminum salt adjuvant (for example, an aluminum hydroxide and / or an aluminum phosphate). The composition of claim 13, wherein the viruses used as source of antigens are cultured in cell culture (e.g., Vero cells) and the composition is free of ovalbumin and ovomucoid. The composition of any one of claims 10 to 12, comprising: fragmented virion antigen or purified surface antigen and an oil-in-water submicron adjuvant emulsion. The composition of claim 10 or claim 15, wherein the emulsion comprises squalene. The composition of claim 16, wherein the composition contains 7.5pg of HA per clade of H5 or 3.75pg of HA per clade of H5. The composition of any one of claims 8-17, which is a 2-valent vaccine composition. 4